Structure-activity relationship studies of the chromosome segregation inhibitor, Incentrom A

Article abstract of DOI:10.1016/j.bmcl.2008.07.006

A series of Incentrom A analogs that inhibit the chromosome segregation process in yeast were synthesized and tested for their effects on chromosome stability and cell proliferation. Pharmacophore and structure-activity relationship of Incentrom A for the anti-yeast activity were established.

Full text of DOI:10.1016/j.bmcl.2008.07.006

Bioorganic & Medicinal Chemistry Letters 18 (2008) 4670–4674
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Structure–activity relationship studies of the chromosome segregation
inhibitor, Incentrom A
a
a
b
b
b,
*
Hee-Yoon Lee a, , Yongsik Jung , Wonyeob Kim , Jin Hee Kim , Min-Soo Suh , Seung Koo Shin
,
*
Hye-Joo Yoon b,
*
a Department of Chemistry and School of Molecular Science (BK21), KAIST, Daejeon 305-701, Republic of Korea
b Bio-Nanotechnology Center, Department of Chemistry, Pohang University of Science and Technology, Pohang, Kyungbuk 790-784, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of Incentrom A analogs that inhibit the chromosome segregation process in yeast were synthe-
sized and tested for their effects on chromosome stability and cell proliferation. Pharmacophore and
structure–activity relationship of Incentrom A for the anti-yeast activity were established.
Ó 2008 Elsevier Ltd. All rights reserved.
Received 8 May 2008
Revised 13 June 2008
Accepted 3 July 2008
Available online 9 July 2008
Keywords:
Inhibition
Chromosome segregation
Yeast
Structure–activity relationship
Pharmacophore
Incentrom A
The process of chromosome segregation is crucial for the con-
servation of normal chromosome number in all eukaryotes. Accu-
rate chromosome segregation requires specialized chromosomal
structures called the centromere (DNA region) and the kinetochore
(protein assembly). The centromeres of the experimental model
yeast Saccharomyces cerevisiae contain specific and absolutely
essential DNA sequences for faithful chromosome segregation,
whereas those of higher organisms have extensive amounts of
repetitive sequences.1,2 Besides, the centromeres of the pathogenic
yeast Candida glabrata, the second most common fungal pathogen
in humans, are closely related to the S. cerevisiae centromeres.3
Thus, small molecules that perturb centromere functions in S. cere-
visiae could be valuable in identifying yeast-specific growth inhib-
itors to lay the basis for the development of novel antifungal
drugs.4 Incentrom A is one such compound.4 To probe the molecu-
lar target of Incentrom A and eventually develop agents which
selectively block the growth of fungi, we studied the pharmaco-
phore and structure–activity relationship (SAR) of Incentrom A
and obtained a compound for photo-affinity labeling.5
measured a minimum inhibitory concentration (MIC) for each
compound.6
N
OH
N
N
Incentrom A (1)
N
N
H
N
a, b
OH
N
R1 R2
R1 R2
N
N
H
N
c, d
Incentrom A (1) has several pharmacophoric units as the two
aromatic moieties are attached to the piperazine ring through alkyl
chains. To establish the structure–activity relationship for Incen-
trom A, we synthesized a series of Incentrom A derivatives and
R1
R2
+
Br
Br
n
n
N
n=1, 2, 3, 4, 6
R1 R2
Scheme 1. Reagents and conditions: (a) epibromohydrin, Cs2CO3, DMF, 60 °C, 15 h;
(b) trans-1-cinnamylpiperazine, THF, EtOH, 60 °C, 1 day. (c) TBAB, benzene, 50% aq.
NaOH, rt, 1 day; (d) trans-1-cinnamylpiperazine, TBAB, CH2Cl2, 50% aq. NaOH, rt, 1
day.
* Corresponding authors. Tel.: +82 42 869 2835; fax: +82 42 869 8370 (H.-Y. Lee).
E-mail address: leeheeyoon@kaist.ac.kr (H.-Y. Lee).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.07.006

H.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4670–4674
4671
Table 1 (continued)
Table 1
Yeast growth inhibitory activities for compounds 1–21
Compound
A
B
MIC (lM)
N
N
HCl
A
N
N
O
17
10–11
>45
B
2
2
2
2
2
Compound
A
B
MIC (
15
lM)
OH
18
19
20
21
Incentrom A(1)
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
Carbazole
OH
3-Nitrocarbazole
5–6
2
14–15
15–16
18–19
>45
OH
3-Acetamidocarbazole
3-Benzamidocarbazole
>45
3
OAc
OBz
>45
4
First, we examined the effects of functional groups and chain length
between piperazine and carbazole nitrogens on the activity as well
5
Table 2
Yeast growth inhibitory activities for compounds 26–36
O
6
>45
R
N
N3
N
HCl
7
>45
N
Compound
R
MIC(lM)
8
12–13
5–6
Ph
Ph
26
7–9
O
9
2
3
4
27
28
29
30a
31
32a
33
34
35
12–14
18–20
>45
OH
Ph
Ph
10
11
12
13
14
15
16
6
OAc
OBz
7–8
20
Ph
44–45
>45
6
30
OH
N
>45
Ph
OH
OH
OH
9–11
5–6
4–5
5–6
Ph
N
45
Ph
Ph
Ph
Ph
2
3
N
25–26
7–9
N
O
36
a
2-Hydroxypropyl chain in place of butyl chain between carbazole and pipera-
zine rings.

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H.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4670–4674
COOEt
OH
N
N
COOH
a-c
Br
j-m
a-i
f-i
N
N
Boc
N
H
OH
52
49
50
22
24
N
N
N
OH
j-l
m, n
j, r, s
h, n-q, o
Br
N
N
Boc
N
H
N
COOEt
51
23
25
Scheme 2. Reagents and conditions: (a) (trimethylsilyl)diazomethane, benzene,
MeOH, rt, 1 h; (b) di-tert-butyl dicarbonate, DIPEA, DMAP, MeOH, rt, 16 h; (c) DIBAL,
THF, 0 °C to rt, 100 min; (d) MsCl, Et3N, CH2Cl2, 0 °C, 30 min; (e) LiBr, acetone,
reflux, 12 h; (f) carbazole, Cs2CO3, DMF, 70 °C, 1 day; (g) TFA, CH2Cl2, rt, 45 min; (h)
trans-cinnamaldehyde, AcOH, MeOH, rt, 1 h; (i) NaBH(OAc)3, rt, 1 day. (j) di-tert-
butyl dicarbonate, Et3N, CH2Cl2, rt, 16 h; (k) oxalyl chloride, DMSO, Et3N, CH2Cl2,
À78 °C to rt, 5 h; (l) diethyl bezylphosphonate, NaH, DMF, 0 °C to rt, 17 h; (m) TFA,
CH2Cl2, rt, 1 h; (n) N-(4-bromobutyl)carbazole, TBAB, CH2Cl2, 50% aq. NaOH, rt, 1
day.
Scheme 3. Reagents and conditions: (a) TBSCl, NaH, THF, 0 °C to reflux, 3 days; (b)
MsCl, Et3N, CH2Cl2, 0 °C, 20 min; (c) diethylmalonate, NaH, TBAI, THF, 0 °C to rt, 1
day; (d) NaCl, DMF, reflux, 38 h; (e) TBAF, THF, rt, 90 min; (f) MsCl, Et3N, CH2Cl2,
0 °C, 20 min; (g) LiBr, acetone, reflux, 9 h; (h) PPh3, CH3CN, reflux, 1 day; (i)
benzaldehyde, t-BuOH, benzene, 70 °C, 10 h; (j) LiAlH4, THF, 0 °C, 20 min; (k) MsCl,
Et3N, CH2Cl2, 0 °C, 20 min; (l) LiBr, Acetone, reflux, 4 h; (m) carbazole, Cs2CO3, DMF,
80 °C, 14 h; (n) 1,4-cyclohexanedione monoethylene acetal, n-BuLi, THF, À78 °C to
rt; (o) Pd/C, H2(1 atm), ethylacetate, 7 h, rt; (p) 1 N HCl, acetone, 50 °C, 1 h; (q)
(carbethoxymethylene) triphenylphosphorane, benzene, reflux, 3 days; (r) PCC,
CH2Cl2, rt, 1 h; (s) diethyl benzylphosphonate, NaH, DMF, 0 °C to rt, 19 h.
as the importance of the carbazole ring in 1. These analogs were
readily synthesized from amine, monosubstituted piperazine and
dibromoalkanes (or epibromohydrin) (Scheme 1).7 The cell prolifer-
ation inhibitory activities of compounds 1–21 are summarized in
Table 1.
Compared to racemic Incentrom A (1), separately prepared
enantiomers (2, 3) did not show any changes in activity, nor did
the protection of the hydroxyl group (4). However, a group bigger
than hydroxyl (5, 7) or the sp2 center in the chain (6) was not tol-
erable for activity. Actually, the hydroxyl group was not necessary
because the compound without hydroxyl group improved the
activity (8). Moreover, the butyl and pentyl chains showed better
activities than other chain lengths (9–12), suggesting that the opti-
mal moiety between carbazole and piperazine rings was a 4-car-
bon chain with no substituent in the middle.
attachment of carbazole produced 24. Compound 25 was prepared
from cyclohexanedione monoethyleneketal. A butylcarbazole unit
was introduced through Wittig olefination followed by hydrogena-
tion of the olefin product. After deprotection of the ketal, two-car-
bon extension was achieved using Wittig olefination reductions,
thus leading to the trans isomer as the major form of disubstituted
cyclohexane.
Finally, the cinnamyl unit was introduced in the same manner
as in the synthesis of 24.
The activities of these compounds were unusual: Both 22 and
23 exhibited undiminished inhibitory activities (MIC of 11–13
and 4–5 lM, respectively), whereas neither 24 nor 25 showed
When indole ring was introduced in place of carbazole (13) or
the rigid carbazole ring was dismantled (14), the inhibitory activity
was completely lost. On the other hand, ring expansion (16) or par-
tially hydrogenated carbazole was tolerated (15, 17), whereas an
introduction of the electron-withdrawing spacer was not (18). Of
the substituted carbazoles (19–21), only the parent 3-nitrocarba-
zole improved the inhibitory activity.
Based on these results, we kept carbazole, fixed the C4 chain
length between carbazole and piperazine rings, and explored the
other parts of Incentrom A to place a photo-affinity tag.
We prepared piperidine analogs of Incentrom A to test the
importance of two nitrogen atoms. Compound 22 was prepared
from 4-piperidinyl butanoic acid (49) by converting 49 into the
corresponding bromide (50) and by attaching carbazole and cin-
namyl groups to 49. The other piperidine analog 23 was prepared
from 4-piperidinyl ethanol (51) as shown in Scheme 2. All carbon
analogs (24, 25) were prepared as shown in Scheme 3. The selec-
tive protection of 52 followed by two carbon extension on one side
using malonate ester synthesis produced a butyl chain for attach-
ment of a carbazole unit and the other ethyl alcohol was extended
to the cinnamyl group through Wittig olefination reaction. Final
inhibitory activity. These results manifested that the presence of
nitrogen in the molecule was crucial for the inhibitory activity.
However, the position of the nitrogen atom was not critical. The
nitrogen atom could play a role either in establishing electrostatic
interaction with target proteins or in retaining the solubility in
aqueous solution or both.
Next, we varied the cinnamyl group. An epoxide analog 26 was
prepared similarly to the preparation of 9 using epoxycinnamyl
bromide rather than cinnamyl bromide. Hydrogenolysis of 26 pro-
duced 27 and its ester analogs 28 and 29 were prepared from 27.
Hydrogenation of the double bond of 9 produced 30. Conforma-
tionally restricted naphthyl compound (31), compounds with vary-
ing chain length (compounds 32-35), and fully saturated
cyclohexyl compound (36) were also prepared, and their activities
are summarized in Table 2. An introduction of epoxide in place of
olefin (26) slightly improved the activity, and the opening of epox-
ide to alcohol (27) retained the activity. The hydroxyl group offered
a site for attaching other functional groups because the acetate
form 28 was still active. However, the activity was lost when a lar-
ger group was attached (29). The saturation of the double bond of
cinnamyl group (30) slightly reduced the activity.

H.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4670–4674
4673
Conformationally restricted naphthyl group (31) significantly
lowered the activity. The benzyl group (32) completely shut off
the activity. The extension of the chain length (33–35) gradually
restored the activity. These results indicated that a flexible alkyl
chain could help folding the compound around the parent pipera-
zine ring. Nonetheless, the presence of benzene ring was not cru-
cial as the cyclohexyl ring (36) showed better activity than 9.
Thus, these Incentrom A analogs could pose a sizable hydrophobic
binding motif. Further optimization of the chain length and attach-
ment of larger hydrophobic groups could help provide insights into
the size of the hydrophobic binding pocket.
Finally, to establish a proper site for attaching affinity tag, we
substituted the phenyl ring of 9. The results are summarized in Ta-
ble 3. The polar phenolic group at the para and meta positions was
well tolerated (37, 42). The activity was improved when phenolic
OH was replaced with OMe. The methoxy substitution at any posi-
tion of the phenyl ring did not change the activity (38, 43, 44).
Whenthe para-methoxygroup was extendedfurtherwith a long-
eralkylchainorbenzylgroup, theactivitywascompletelylost. These
results indicated that this binding site could tolerate hydrophobic
groups but only in a limited space. On the other hand, the activity
got improved (45, 46) when phenolic OH was replaced with an
amine functional group. Although the dimethyl substitution of the
amine quenched the activity, an introduction of an azide functional
group showed an excellent activity (48). Since the azide group is a
good candidate for photo-affinity labeling, compound 48 could serve
as the probe for identifying the target protein of Incentrom A.
Compounds 46 and 48 were readily prepared from p-aminocin-
namaldehyde in a few steps (Scheme 4). Reductive amination pro-
vided 46 and diazotization followed by azide replacement
produced 48. This synthetic scheme provided enough material
for the photo-affinity labeling study and allowed synthesis of deu-
terated compound for mass spectrometric identification of target
proteins.8 Both in vitro and in vivo identification of target proteins
are under way. In addition, the chromosome missegregation activ-
ities of Incentrom A (1) and its important analogs, 9 and 48, are
shown in Fig. 1.9 As evidenced by a dramatic increase in red colo-
nies, these compounds significantly induced a tester chromosome
loss at much lower concentration than Incentrom A.
Table 3
Yeast growth inhibitory activities for compounds 37–48
N
N
N
HCl
R
Compound
R
MIC (lM)
37
38
39
40
41
42
43
44
45
46
47
48
p-OH
p-OMe
p-OBn
28–29
8–9
>45
>45
>45
27–28
12–13
12–14
16–17
15–16
42–44
5
p-OC6H13
p-OC8H17
m-OHa
m-OMea
m-OMea
p-NH2
In summary, we established the pharmacophore of Incentrom
A: The carbazole unit with four carbon tether was optimal for
the inhibitory activity (9), and at least one nitrogen atom of the
piperazine ring with a long hydrophobic chain was essential. We
a
p-NH2
a
p-NMe2
a
p-N3
a
These substituents were attached on the cinnamyl group.
also identified
a
phenylazide containing compound 48 as a
N
N
C
HO
N
N
N
N
N
a-c
d, e
+
N
N
H
NHBoc
NH2
N3
46
48
Scheme 4. Reagents and conditions: (a) AcOH, MeOH, rt, 2 h; (b) NaBH(OAc)3, rt, 1 day; (c) TFA, CH2Cl2, rt, 45 min; (d) conc. HCl, NaNO2, 0 °C, 15 min; (e) NaN3, 0 °C, 20 min.
Figure 1. Chromosome missegregation activities of Incentrom A analogs.

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H.-Y. Lee et al. / Bioorg. Med. Chem. Lett. 18 (2008) 4670–4674
6. The MIC was defined as the lowest drug concentration at which 95–100%
inhibition of yeast viability (YPH278 strain) was observed in comparison to the
DMSO control. The 10 mM stock solution of each compound was prepared in
DMSO. Yeast cells (ꢀ6.6 Â 105 cells/mL) were treated with DMSO or Incentrom
A analogs and grown for 24 h at 30 °C. Appropriate dilutions of each culture
were plated on YPD medium. Yeast colonies were counted to calculate%
reduction in yeast viability. The number of viable cells obtained from chemical-
treated culture was normalized to the one from DMSO culture. Each MIC
represents a mean or a range of values from 2–4 independent experiments.
7. (a) Bombrun, A.; Gerber, P.; Casi, G.; Terradillos, O.; Antonsson, B.; Halazy, S.
J. Med. Chem. 2003, 46, 4365; (b) Li, X.; Mintz, E. A.; Bu, X. R.; Xehnder, O.;
Bosshard, C.; Günter, P. Tetrahedron 2000, 56, 5785.
photo-affinity probe to isolate target proteins in vivo. The present
structure–activity relationship study provided further insights into
the rational design of Incentrom A analogs.
Acknowledgment
This work was supported by KOSEF (Grant R01-2005-000-
11019-0).
References
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carrying a tester minichromosome was grown in 5 mL of selective medium to
log phase (OD660 = 2). Subsequently ꢀ10 generations of non-selective growth
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were initiated by inoculating 20 mL of YPD with 20 lL of the selectively grown
culture and treating with various compounds or DMSO. During the non-
selective growth period, yeast cells that lost a tester chromosome produce red-
colored colonies.