Part II. Development of novel colchicine-derived immunosuppressants with improved pharmacokinetic properties

Article abstract of DOI:10.1016/j.bmcl.2012.08.068

We have developed a new series of immunosuppressant with improved pharmacokinetic properties as the second-generation of colchicine analogs, which were designed based on the privileged structure derived from our previous work. In particular, we identified an analog (14), which exhibited a potent in vitro activity (IC₅₀: 5 nM) in MLR and excellent in vivo efficacy in the Zymosan A-induced arthritis model, in the Carrageenan-induced edema model and in the local lymph node assay (LLNA). Analog 14 also revealed a good oral bioavailability (F: 67.3%) in BALB/c mice.

Full text of DOI:10.1016/j.bmcl.2012.08.068

Bioorganic & Medicinal Chemistry Letters 22 (2012) 6750–6755
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Part II. Development of novel colchicine-derived immunosuppressants
with improved pharmacokinetic properties
Dong-Jo Chang a, , Sujin Lee a, Jaebong Jang a, Soon-Ok Kim b, Wan-Joo Kim b, Young-Ger Suh a,
⇑
⇑
a College of Pharmacy, Seoul National University, 1 Gwanak-ro, Gwanak-gu, Seoul 151-742, Republic of Korea
b Team of Biotechnology and Drug Discovery, Chem-Tech Research Incorporation, 778-1 Ilpae Dong, Namyangju City, Gyeonggi-do 472-930, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
We have developed a new series of immunosuppressant with improved pharmacokinetic properties as
the second-generation of colchicine analogs, which were designed based on the privileged structure
derived from our previous work. In particular, we identified an analog (14), which exhibited a potent
in vitro activity (IC50: 5 nM) in MLR and excellent in vivo efficacy in the Zymosan A-induced arthritis
model, in the Carrageenan-induced edema model and in the local lymph node assay (LLNA). Analog 14
also revealed a good oral bioavailability (F: 67.3%) in BALB/c mice.
Received 30 July 2012
Revised 15 August 2012
Accepted 17 August 2012
Available online 25 August 2012
Keywords:
Autoimmune disease
Colchicine
Ó 2012 Elsevier Ltd. All rights reserved.
Cyclosporine A (CsA)
Immunosuppressant
Mixed-lymphocyte reaction (MLR)
Rejection of organ-transplantation
Since the first organ-transplantation between human twins was
successfully performed in 1954,1 a large number of patients have
received organ-transplants every year. However, they should be
consistently treated with the immunosuppressive drugs to over-
come rejection by their own immune system.2 Generally, rejec-
tions caused by organ-transplantation and autoimmune diseases
including rheumatoid arthritis, psoriasis, autoimmune pancreatitis
and multiple sclerosis are mainly mediated by T cells with an assis-
tance of B cells affecting the diseases through antibody produc-
tion.3 The T cell-mediated immune diseases are treated with a
available immunosuppressant for organ-transplantation and treat-
ment of autoimmune diseases still remains.
Recently, we reported a colchicine-derived compound (2, Fig. 1),
which exhibited strong immunosuppressive activities in the
in vitro and in vivo models.14 However, this lead compound turned
out to have poor pharmacokinetic profiles including seriously poor
bioavailability. The poor pharmacokinetic properties were likely
due to the extremely low solubility in water. Thus, increase of
aqueous solubility was considered for the improved oral bioavail-
ability. The metabolically labile nitrate group seemed also to affect
the bioavailability. We herein describe our recent effort to discover
the second-generation of colchicine analogs that have the im-
proved pharmacokinetic properties with maintaining the excellent
immunosuppressive activity.
As shown in our preliminary work for the improved pharmaco-
kinetics (Table 1), analog 4 possessing the thiomethoxy (R1) and N-
4-(nitrooxy)butanoyl (R2) groups exhibited a threefold lower solu-
bility in the media containing 20% of serum and a 10-fold higher
activity compared to colchicine possessing the methoxy (R1) and
N-acetyl (R2) groups. Analog 5 possessing the thiomethoxy (R1)
and 3-(nitrooxymethyl)benzoyl (R2) groups showed a lower solu-
bility and a higher activity compared to analog 4. Thus, we as-
sumed that the lower solubility of 5, compared to those of
colchicine and 4, was due to a stacking effect of aryl moiety in
the N-side chain of 5. Based on our previous structure–activity
relationship and the present structure–solubility relationship
shown in Table 1, we designed the new analogs for optimization
of the N-side chain of 2 as described in Figure 2. Our strategy
variety of therapies including medication of cyclosporine
A
(CsA)4,5 and FK506.6,7 Antibody therapy reduces a specific group
of cells such as T cells or B cells and alter signal transduction path-
ways using effector cells.8,9 CsA as one of the best immunosuppres-
sant acts on calcineurin and selectively induces an inhibition of T
cell proliferations.10 Despite a great deal of therapeutic applica-
tions of immunosuppressant for organ-transplantation and auto-
immune diseases, there still remains a need for more effective
and less toxic immunosuppressant. Especially, many current
immunosuppressive drugs revealed serious side effects such as re-
nal toxicity, neurotoxicity and hypertension.11–13 In addition, the
most widely prescribed immunosuppressive drugs containing
CsA or FK506 are not orally available due to high molecular weight
and lipophilicity. Thus, a strong demand for the safe and orally
⇑
Corresponding authors. Tel.: +82 2 880 7875; fax: +82 2 888 0649.
E-mail address: ygsuh@snu.ac.kr (Y.-G. Suh).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.08.068

D.-J. Chang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6750–6755
6751
Cl
ONO2
HO
O
O
O
H
N
H
O
O
MeO
MeO
N
N
N
N
N
NH
O
O
O
O
O
OMe
N
O
H
N
H
N
N
H
N
SMe
O
O
2
CsA (1)
Figure 1. Structures of cyclosporine A (CsA, 1) and 2.
Table 1
O
O
O
O
b
a
Structure–solubility relationship of the previously reported colchicine derivatives
EtO
EtO
HO
HO
R2
MeO
PO(OEt)2
NH
15
17
19
16
MeO
OMe
O
, colchicine, R1 = OMe, R2 = acetyl
b
c
3
O
15
4, R1 = SMe, R2 = 4-(nitrooxy)butanoyl
EtO
R1
5, R1 =SMe, R2 = 3-(nitrooxymehyl)benzoyl
Compound
Solubilitya
(l
M)
Activityb (IC50, nM)
18
3, Colchicine
300
100
10
2300
210
68
O
O
O
b
d
4
5
2
HO
EtO
EtO
c
—
8
20
21
22
a
Solubility of each compound was determined by evaluating both turbidity and
light scattering using a UV/visible spectrophotometer and a Nepheloskan instru-
ment. Solubility was assessed at 37 °C in the dosing media, which contained 20%
serum.
Scheme 2. Reagents and conditions: (a) (CH2O)n, NaH, DMF, rt, 66%; (b) NaOH,
MeOH, H2O, rt, 17: 83%, 19: 78%, 22: 69%; (c) methacrolein, NaH, DMSO, rt, 91%; (d)
(CH2O)n, NaH, DMF, rt, 91%.
b
MLR assay. IC50 values are mean of three experiments, standard deviation below
20%.
c
Solubility of 2 was not determined.
designed alternative analogs avoiding the nitrate group, which is
metabolically labile15,16 because analog 2 devoid of the nitrate
involving incorporation of an aliphatic side chain, which consisted
of alkenes or three-membered ring systems with sp2 or relevant
hybridizations, envisaged elevations of both solubility and activity.
However, our initial analogs possessing the five- or six-membered
heterocycles including pyrrole, thiophene, pyridine and pyrimidine
instead of the 2-chlorobenzene moiety exhibited poor immuno-
suppressive activities (IC50: >500 nM, data not shown). Thus, we
group exhibited an excellent immunosuppressive activity (IC50
22 nM).
:
As depicted in Scheme 1, most of the designed compounds were
prepared by the EDC-mediated amide coupling reactions of 7-
deacetylthiocolchicine 6 with various aliphatic carboxylic acids.14
Some aliphatic carboxylic acids were synthesized from triethyl
2-phosphonobutyrate 15 or 3-butenoic acid 20 employing
Cl
ONO2
Aliphatic side chain
for solubility
O
O
MeO
MeO
MeO
NH
NH
MeO
Increase of Solubility
sp2 or relevant hybridization
for activity
OMe
Maintenance of Activity
OMe
2
O
O
SMe
SMe
Figure 2. Strategy for design of the pharmacokinetically improved analogs.
O
O
1
R1
NH
MeO
MeO
MeO
MeO
7, R1=
11
, R =
NH2
O
a
O
O
O
O
O
1
8
, R =
12, R1=
OMe
OMe
O
SMe
9, R1=
13
SMe
1
. R =
6
1
O
10,
R =
14, R1=
Scheme 1. Reagents and conditions: (a) carboxylic acids, EDC, CH2Cl2, rt, 7: 92%,8: 96%, 9: 78%, 10: 62%, 11: 63%, 12: 53%, 13: 71%, 14: 98%.

6752
D.-J. Chang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6750–6755
O
O
MeO
MeO
MeO
MeO
O
NH
NH
a
OMe
OMe
O
O
SMe
SMe
7
23
HO
O
HO
HO
O
b
c
O
O
NH NH
Cbz
O
HO
HN Cbz
HO
NH2
24
O
O
25
SMe
26
MsO
O
O
O
O
O
O
e
d
N
NH NH
Cbz
NH
Cbz
O
O
O
O
SMe
SMe
27
28
O
O
O
O
O
f
g
NH
NH
N
NH
O
O
O
O
O
SMe
SMe
29
30
Scheme 3. Reaction conditions and reagents: (a) mCPBA, CH2Cl2, rt, 82%; (b) CbzCl, NaHCO3, THF, water, rt, 93%; (c) 6, EEDQ, Et3N, CH3CN, 48%; (d) MsCl, Et3N, CH2Cl2, rt,
quant.; (e) t-BuOK, t-BuOH, CH2Cl2, 0 °C to rt, 38%; (f) KOH, THF, water, rt, 9%; (g) MeI, K2CO3, MeOH, 50 °C, 65%. EEDQ = N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.
O
MeO
MeO
MeO
MeO
NH2
O
NH
a
Table 2
OMe
OMe
Immunosuppressive activities of the prepared analogs in MLR
O
R1
NH
OMe
OMe
MeO
31
32
O
O
MeO
MeO
MeO
MeO
MeO
OMe
NH
NH
b or c or d
O
R2
OMe
OMe
O
b
Compounda
R1
—
R2
IC50 (nM)
O
SMe
R
CsA
2
7
8
9
10
11
12
13
14
Carbonyl
23
29
30
32
Carbonyl
33
Carbonyl
34
—
30
8
67
14
33, R = SOMe
34
2-Chloro-3-nitrooxy methyl benzoyl
2-Butenoyl
3-Butenoyl
3-Pentenoyl
2,4-Hexadienoyl
2-Methylenebutanoyl
2-Methylene-3-butenoyl
2-Ethyl-4-methyl penta-2,4-dienoyl
2-Methylcyclopropane
SMe
3-Methyloxirane-2-carbonyl
3-Methylaziridine-2-carbonyl
1,3-Dimethylaziridine-2-carbonyl
2-Methylcyclopropane
OMe
2-Methylcyclopropane
SOMe
2-Methylcyclopropane
SO2Me
2-Methylcyclopropane
H
2-Methylcyclopropane
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
SMe
, R = SO2Me
35, R = H
702
1670
4
Scheme 4. Reagents and conditions: (a) 2-methylcyclopropanecarboxylic acid,
EDC, CH2Cl2, rt, 71%; (b) R = SOMe:mCPBA (1.05 equiv), CH2Cl2, rt, 82%; (c)
R = SO2Me:mCPBA (2.5 equiv), CH2Cl2, rt, 84%; (d) R = H, (i) Raney Ni, EtOH, rt,
70%, (ii) Pd/C, toluene, reflux, 31%.
86
112
31
O
5
MeO
MeO
MeO
MeO
SMe
SMe
SMe
119
46
38
NH2
O
NH
a
OMe
OMe
O
1247
N.A.
N.A.
N.A.
N.A.
OMe
OMe
36
37
O
MeO
MeO
Carbonyl
35
Carbonyl
38
NH
b
OMe
O
Carbonyl
SMe
38
a
All compounds were purified by column chromatography and then recrystal-
lization (>95%).
Scheme 5. Reagents and conditions: (a) 2-methylcyclopropanecarboxylic acid,
EDC, CH2Cl2, rt, 86%; (b) NaSMe, THF, H2O, 50 °C, 59%.
b
IC50 values are mean of three experiments, standard deviation below 20%.

D.-J. Chang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6750–6755
6753
Table 3
Mixed-lymphocyte reaction (MLR) assay on splenocytes har-
vested from C57BL/6 and BALB/c mice was conducted to explore
immunosuppressive activities of the prepared analogs as shown
in Table 2.22 Among the analogs with an alkene group, analog 10
possessing the 2,4-hexadienoyl substituent exhibited the most po-
tent activity (4 nM), which is twofold more potent compared to the
parent compound 2. Generally, the analogs with a Michael accep-
tor such as 7, 10, 11 and 13showed excellent immunosuppressive
activities with IC50s less than 100 nM. Interestingly, analogs 14–30
possessing the three-membered rings, of which sp3-hybridization
is known to have sp2-characters,23 revealed good or excellent
immunosuppressive activities. In particular, analog 14 with a
cyclopropane exhibited highly potent immunosuppressive activity
with an IC50 of 5 nM. Replacement of the thiomethoxy substituent
with methoxy, sulfinyl or sulfonyl group resulted in significant de-
crease (32) or loss (33, 34) of activity as our previous report.14 Re-
moval of the thiomethoxy group (35) eliminated the activity,
which supported a crucial role of the thiomethoxy group for immu-
nosuppressive activity of the analogs. In addition, no immunosup-
pressive activity of the antipode (38) of 14 revealed the important
stereochemical feature of the tested analogs.
Analogs 10 and 14, which were most potent in MLR assay, were
further evaluated using the lymphoproliferation assay on the B and
T lymphocyte-activated cells.24 Both analogs exhibited good sup-
pressive activities against the activated B cells than T cells as
shown Table 3. Analog 14 was finally selected because it exhibited
higher potency in suppression of the activated T cells, which is
important for treatment of the rejection in organ-transplantation
and autoimmune diseases. Thus, we further evaluated analog 14
using the cell viability test on the cell lines including naive
lymphocytes, activated lymphocytes, bone marrow-derived den-
dritic cells (BM-DC) and bone marrow-derived macrophages
(BM-Mac). As shown in Figure 3, analog 14 exhibited potent inhib-
itory activity against activated lymphocytes while it did not affect
Lymphoproliferation assay on T and B lymphocyte-activated cells
Compound
B cells (IC50, ng/mL)a
T cells (IC50, ng/mL)a
10
14
5.7
5.9
40.7
31.0
a
IC50 values are mean of three experiments, standard deviation below 20%.
Horner–Wadsworth–Emmons olefination or aldol condensation,
followed by hydrolysis of the resulting ester as shown in Scheme 2.
Synthesis of the analogs possessing the 3-membered heterocycles
such as epoxide and aziridine are outlined in Scheme 3. Epoxida-
tion of 7 with mCPBA produced epoxide 23 as a diastereomeric
mixture in good yield. Cbz-protection of -threonine followed by
L
amide coupling reaction in the presence of EEDQ and Et3N afforded
26,17 which was subsequently reacted with methanesulfonyl chlo-
ride to give 27. Intramolecular cyclization of the Cbz-protected
amine 27 in the presence of t-BuOK produced aziridine 28, which
was subjected to Cbz-deprotection and methylation to give 30.
We also introduced methoxy, sulfinyl and sulfonyl group instead
of the thiomethoxy group of 2 because the sulfide group was likely
to be easily oxidized via the phase I metabolism.18 As shown in
Scheme 4, oxidation of 14 using mCPBA produced the sulfoxide
analog33 and the sulfonyl analog 34, respectively. Analog 35 de-
void of the thiomethoxy group was prepared via a sequence of Ra-
ney Ni-mediated reduction and subsequent aromatization in the
presence of Pd/C.19 Finally, analog 38, which lately turned out to
be the best analog in terms of in vitro and in vivo activities, was
prepared from 36, an antipode of 31, for analysis of the stereo-
chemical effects of the N-side chain of 14. Analog 38 was easily
prepared from (+)-N-deacetylcolchicine 3620,21 by an EDC-medi-
ated amide coupling reaction and NaSMe treatment as shown in
Scheme 5.
Figure 3. Cell viability of analog 14. (a) Cell viability on naive lymphocytes, (b) cell viability on activated lymphocytes, (c) cell viability on bone marrow-derived dendritic
cells, (d) cell viability on bone marrow-derived macrophages.

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D.-J. Chang et al. / Bioorg. Med. Chem. Lett. 22 (2012) 6750–6755
Figure 4. In vivo test of analog 14. (a) Effect on Zymosan A-induced arthritis, (b) effect on Carrageenan-induced paw edema, (c) local lymph node assay (LLNA) induced by
DNCB.
other cell lines, suggesting that analog 14 is much less toxic in nor-
mal cells.
benzene (DNCB).27,28 Figure 4c also revealed that analog 14 effec-
tively decreased the lymph node increased by DNCB. As
mentioned in the introduction part, the final goal of our work fo-
cused on the discovery of immunosuppressive agents with im-
proved pharmacokinetic profiles with the immunosuppressive
activity maintained. Thus, we determined PK properties of analog
14. As shown in Figure 5, analog 14 revealed systemic clearance
In vivo immunosuppressive effect of 14was evaluated using the
arthritis, edema model and the local lymph node assay (LLNA). The
C57BL/6 mice was treated with 14 after subcutaneous injection of
Zymosan A (50 ml of 3 mg/ml) from Saccharomyces cereviciae (Sig-
ma, St. Louis, MO) into the hind footpad.25,26 As well, the C57BL/6
mice was treated with 14 after a paw edema was induced by a sub-
cutaneous injection of 0.1 ml of 1% Carrageenan into the left hind
paw.25 Figure 4a and b illustrated that analog 14 reduced both
arthritis and edema in a dose dependent manners. Considering that
above in vivo models are mainly associated with activation of mac-
rophages, we tested 14 using the in vivo local lymph node assay
(LLNA), which envisioned an in vivo functional assay for immune
reactivity to the T cell-dependent antigen like 2,4-dinitrochloro-
(CL) with a half value of hepatic blood flow and a half-life (t1/2
)
of 3 h, which seemed suitable for oral administration. Furthermore,
analog 14 exhibited good oral bioavailability (F: 67.3%), which
envisioned a good possibility for oral administration.
In conclusion, we developed immunosuppressive agents with
improved pharmacokinetic properties and strong immunosuppres-
sive activity based on the structure–activity and structure–solubility
relationships of 2, which was identified as an immunosuppressive
agent via our previous work. In particular, the alkene or three-
membered ring-incorporated analogs 10 and 14 exhibited
improved in vitro activities. In addition, analog 14 exhibited a
good efficacy in both in vivo models mediated with T cell and
macrophages. Analog 14 exhibited promising pharmacokinetic
properties with bioavailability over 67%, which was significantly
improved compared to the parent compound 2. Currently, further
studies on 14 including in vivo transplantation experiments are in
progress.
Acknowledgement
We are grateful to the KT & G Corp. for financial support of this
research.
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Figure 5. Pharmacokinetics of analog 14 in BALB/c mice.

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