Synthesis and SAR of sulfonyl- and phosphoryl amidine compounds as anti-resorptive agents

Article abstract of DOI:10.1016/j.bmcl.2009.11.104

Sulfonyl amidines (1) and phosphoryl amidines (2), which were efficiently synthesized via a Cu-catalyzed one pot reaction, showed potent anti-bone resorptive activity in vitro. Structure activity relationship studies led to the identification of numerous

Full text of DOI:10.1016/j.bmcl.2009.11.104

Bioorganic & Medicinal Chemistry Letters 20 (2010) 541–545
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and SAR of sulfonyl- and phosphoryl amidine compounds
as anti-resorptive agents
Myung Yun Lee a,d, , Myung Hee Kim b, , Jinho Kim c, Seok Hwan Kim c, Bum Tae Kim d, In Howa Jeong a,
Sukbok Chang c, Seong Hwan Kim b, Sung-Youn Chang d,
*
a Department of Chemistry and Medical Chemistry, Yonsei University, Wonju 220-710, Republic of Korea
b Laboratory of Chemical Genomics, Pharmacology Research Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Sinseongno19, Yuseong-gu,
Daejeon 305-600, Republic of Korea
c Department of Chemistry, Korea Advanced Institute of Science & Technology (KAIST), Guseong-dong, Yuseong-gu, Daejeon 305-701, Republic of Korea
d Medicinal Chemistry Research Center, Bio-Organic Science Division, Korea Research Institute of Chemical Technology, Sinseongno19, Yuseong-gu, Daejeon 305-600, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Sulfonyl amidines (1) and phosphoryl amidines (2), which were efficiently synthesized via a Cu-catalyzed
one pot reaction, showed potent anti-bone resorptive activity in vitro. Structure activity relationship
studies led to the identification of numerous osteoclast differentiation inhibitors.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 23 September 2009
Revised 13 November 2009
Accepted 19 November 2009
Available online 26 November 2009
Keywords:
Sulfonyl amidine
Phosphoryl amidine
Osteoporosis
Osteoclast
Anti-resorptive agent
A practical synthesis of amidines would be very helpful for
medicinal chemists because amidines are found in many bioactive
natural products1 and identified as important pharmacophores.2
Recently, efficient one-pot syntheses of amidines using Cu-cata-
lyzed three component coupling reactions have been published.3,4
The reaction was proposed to proceed via the formation of a kete-
nimine intermediate, which is generated in situ by the Cu-cata-
lyzed cycloaddition of sulfonyl- or phosphoryl azides with
terminal alkynes followed by the ring-cleavage of the resultant tri-
azoles.5 It is believed that the excellent reactivity of the ketenimine
intermediate allows for the amazingly diverse reactions with pro-
nucleophiles such as amines, alcohols, water, and imidazole. In the
present study, we synthesized various sulfonyl- and phosphoryl
amidines according to the reported procedure and evaluated their
biological activity.
obtained in relatively low yield with Cu-catalyzed one-pot
reaction, was synthesized in 88% yield using substitution of the
phenoxy group with methoxide.4 Subsequent hydrolysis of the
N-dimethoxy phosphoryl amidine (2k) with TMSCl in the presence
of NaI gave the amidine containing phosphonic acid (2l) in 72%
yield8 (Scheme 1).
Based on the methodology in Scheme 1, we prepared 64 sulfo-
nyl amidine derivatives (1) and screened them in vitro for their
anti-cancer, anti-obesity, bone forming, and anti-bone resorptive
activities (Data are not shown). From the screening, amidine 1a
showed anti-resorptive activity with tartrate-resistant acid phos-
phatase (TRAP, a biomarker of osteoclastogenesis; IC50 value of
16.7
did not exhibit efficient anti-cancer, anti-obesity, and bone form-
ing activities at a concentration of 10
M.9 These results suggested
lM in RAW264.7 cells). In contrast, these same derivatives
l
Sulfonyl amidines (1) were synthesized from alkyne, amine, and
sulfonyl azide in the presence of CuI catalyst at rt in 66–99% yield.6
In addition, phosphoryl amidines (2) were synthesized from phos-
phoryl azide instead of sulfonyl azide in 38–82% yield.7 N-Dime-
thoxy phosphoryl amidine (2k), which had been previously
that amidine derivatives could be developed as selective anti-
resorptive osteoporosis drugs.
Bone is constantly remodeled through osteoblast-mediated for-
mation of bone matrix and osteoclast-mediated bone resorption in
order to maintain skeletal strength and integrity. However, an
imbalance in bone remodeling caused by increased bone resorp-
tion over bone formation leads to the reduction of bone mineral
density that is a major cause of several bone disorders such as
osteoporosis.10 Since the loss of bone mass can increase the risk
* Corresponding author. Tel.: +82 42 860 7161; fax: +82 42 861 0307.
E-mail address: sychang@krict.re.kr (S.-Y. Chang).
These authors contributed equally to this Letter.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.11.104

542
M. Y. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 541–545
O
S
CuI(10 mol%)
THF, rt
N
O
S
O
3
N
N
R4
N3
R4
R1
+
R1
O
[Cu]
4
R2
R3
N
H
5
R4
R3
O
R2
O
N
[Cu]
R1
S
O
R4
O
C
N
R1
S
1-4 h
- N2
N
1a-p
CuI(10 mol%)
THF, rt
O
O
N
N
R1
N3
P OPh
+
N
P
OPh
OPh
R1
OPh
[Cu]
4
6
R2
R3
N
H
R3
O
OPh
R2
O
[Cu]
R1
P
N
5
OPh
OPh
OPh
R1
C
N
P
- N2
1-12 h
N
2a-j
i) TMSCl, NaI
CH3CN
50 oC, 4 h
MeONa
N
N
N
O
O
O
OPh
OPh
OMe
OH
OH
THF
rt, 7 h
Ph
Ph
Ph
P
P
ii) H2O
rt, 12 h
P
N
N
N
OMe
2a
2k
Scheme 1. Syntheses of sulfonyl- and phosphoryl amidines.
2l
of fractures, which can lead to serious problems including substan-
tial skeletal deformity, pain, increased mortality and severe eco-
nomic burden,11 the prevention or treatment of loss of bone
mass is an important means of improving the quality of life.
Anti-resorptive agents have been considered as the therapeutic
mainstay for osteoporosis, but there is a need for new anti-resorp-
tive agents without the side effects such as bisphosphonate-related
osteonecrosis of the jaw.12 Therefore, in this report, amidines were
investigated as potential alternative inhibitors of osteoclast
differentiation.
tion (1i) resulted in an inactive analogue. Therefore, aliphatic
chains without polar functionality appear to be the best substitu-
ents for R1.
Interestingly, substitution of the NR2R3 group of inactive deriv-
ative 1d resulted in improved inhibitory activity. Substitution of
the diisopropylamino NR2R3 group with pyrrolidine (1j), piperidine
(1k), and cis-2,6-dimethylpiperidine (1l) gave an improvement in
activity (88%, 57%, and 5% TRAP activity of control at 10
lM,
respectively) as compared to 1d itself (105% of control at 60
lM).
Refinement of amidine compound 1l with n-butyl R1 group (1m)
To identify more efficient anti-resorptive amidine derivatives, a
diverse range of sulfonyl- and phosphoryl amidine derivatives
were synthesized as outlined in Scheme 1 and the structure-activ-
ity relationships (SAR) were investigated (Table 1 and Table 2). The
activity was evaluated using a TRAP activity assay and the data are
presented as % of control sample which received no added test
compounds.13 In cases of the most promising candidates, IC50 val-
ues were also calculated, with compounds tested over the concen-
instead of a 3-chloropropyl group resulted in similar activities
(TRAP IC50 values of 1.8 and 3.3 lM for 1l and 1m, respectively).
Changing the p-tolyl R4 group of sulfonyl amidine 1e into p-nitro-
phenyl (1n), 2-pyridyl (1o), and methyl (1p) groups was not effec-
tive. Thus, in the sulfonyl amidine series, 1l and 1m were the most
potent inhibitors of osteoclast differentiation with IC50 values of
<5
l
M.
In addition to sulfonyl amidines, phosphoryl amidine deriva-
tration range of 0.3–30
l
M.
tives (2a–2l) were synthesized according to Scheme 1 and SAR of
the series were also investigated (Table 2).
The initial test compound 1a containing n-butyl (R1), diisopro-
pylamino (NR2R3), and p-tolyl (R4) groups showed potent anti-
In general, the phosphoryl amidines were more potent than the
osteoclastogenic activity with 3% of control activity at 60
l
M. To
sulfonyl amidines, showing anti-resorptive activity at 10
whereas most of the sulfonyl amidines required concentrations
lM
investigate the effect of the R1 group on the activity, the diisopro-
pylamino and p-tolyl groups were held fixed, and the R1 group was
varied. The bulkier t-butyl analogue (1b) showed similar activity at
as high as 60 lM to exhibit a similar activity (Table 1).
The phosphoryl amidine compound 2a containing phenyl R1,
60
lM, but introducing polar functionalities such as hydroxyl (1c)
diisopropylamino NR2R3, and phenyl R5 showed potent activity at
or chloro groups (1d) reversed the inhibitory activity. The phenyl
derivative 1e did not display a significant improvement in inhibi-
tion, regardless of the presence of the electron donating dimethyl-
amino (1f) or electron withdrawing trifluoromethyl (1g) or nitro
(1h) substituents at the para position. Moreover, pyridyl substitu-
10
l
M, with activity ꢀ8-fold more potent than the control.14 With
the exception of 2b, modification of the R1 group of phosphoryl
amidine 2a, while holding the diisopropylamino NR2R3 and the
phenyl R5 groups constant, resulted in similar activity against
osteoclast differentiation. Introducing the electron-donating

M. Y. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 541–545
543
Table 1
SAR studies of sulfonyl amidines
R3
O
R2
N
O
R4
R1
R3
S
N
R2
R1
R4
Trap activitya (% of control)
N
Compds
1a
3
10
N
1b
N
N
N
N
N
HO
Cl
1c
59
1d
105
98
1e
1f
114
N
N
N
N
1g
1h
105
105
F3C
O2N
1i
1j
107
88b
N
Cl
Cl
N
1k
1l
57b
5b
0
N
Cl
N
1m
N
N
1n
124
NO2
N
1o
1p
84
98
N
N
CH3
a
TRAP activity at 60
TRAP activity at 10
l
M of amidine compounds except 1j, 1k, and 1l.
b
lM of amidine compounds.

544
M. Y. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 541–545
Table 2
SAR studies of phosphoryl amidines
R3
O
R2
N
OR5
OR5
R1
P
N
R2
R3
R1
R5
Trap activitya (% of control)
IC50
2.0
(lM)
N
Compds
2a
Ph
13
N
N
N
2b
2c
Ph
Ph
67
14
21.2
2.6
MeO
O2N
2d
2e
2f
Ph
Ph
Ph
14
0.4
10
nd
N
N
N
N
2.4
7.3
2g
2h
Ph
Ph
24
51
4.2
N
O
N
10.6
But
NH
2i
Ph
Ph
Me
H
27
28
4.8
6.5
nd
nd
NH
2j
2k
90
N
N
2l
107
a
TRAP activity at 10 lM of amidine compounds.
methoxy group at the para-position of the phenyl R1 group (2b)
modestly inhibited the activity (67% of control), but substitution
of the methoxy group by the electron withdrawing NO2 group
(2c) restored the inhibitory activity. The phosphoryl amidine deriv-
atives containing a heteroaromatic ring such as pyridyl (2d), a
cycloalkane such as cyclohexyl (2e), and an alkyl chain such as
n-butyl (2f) groups afforded potent activities comparable to 2a.
The phenyl R1 and R5 groups were then held constant, allowing
the SAR investigation for optimization of the NR2R3. Substitution of
diisopropylamine with piperidine (2g), t-butylamine (2i) and n-
propylamine (2j) was successfully carried out, yielding several po-
tent inhibitors with decreased anti-resorptive activity to ꢀ25% of
control values, but replacement by a morpholine (2h) at this posi-
tion resulted in a twofold decrease in activity to 51% of control.
While keeping phenyl (R1) and diisopropylamino (NR2R3)
groups constant, substitution of the diphenyl phosphonate
(R5 = Ph) group with either dimethyl phosphonate (2k) or phos-
phonic acid (2l) resulted in an inactive inhibitor implying that
the diphenyl phosphonate functionality (R5 = Ph) is essential for
activity. From the phosphoryl amidine series,
showed potent activity with IC50 values of <10 M.
7 compounds
l
Inconclusion, anovelseriesof sulfonyl-andphosphorylamidines
were synthesized efficiently via a Cu-catalyzed one pot reaction. SAR
studies revealed that phosphoryl amidines are more potent than sul-
fonylamidines, and thediphenylphosphonate functionality of phos-
phoryl amidines is critical for anti-resorptive activity. Target protein
identification with an affinity probe and in vivo experiments with
ovariectomized mice are currently underway.

M. Y. Lee et al. / Bioorg. Med. Chem. Lett. 20 (2010) 541–545
545
was diluted by adding CH2Cl2 (2 mL) and aqueous NH4Cl solution (3 mL). The
mixture was stirred for an additional 30 min and two layers were separated.
After the aqueous layer was extracted with CH2Cl2 (3 mL Â 3), the combined
organic layers were dried over MgSO4, filtered, and concentrated in vacuo. The
crude residue was purified by flash column chromatograph with an
appropriate eluting solvent system.
Acknowledgments
This work was supported by a grant from the Korea Healthcare
Technology R&D Project, Ministry of Health, Welfare & Family
Affairs, Republic of Korea (A084242) and Korea Research Founda-
tion Grant (KRF-2005-015-C00248). This research was also sup-
ported by the KOSEF (R01-2007-000-10618-0) and the KRICT (SI-
0905).
7. General procedure for the preparation of phosphoryl amidines (2): For the
synthesis of phosphoryl amidines(2), amine nucleophile (0.75 mmol, 1.5 equiv)
was added to the mixture of azide (0.5 mmol, 1.0 equiv), alkyne (1 mmol,
2.0 equiv), and CuI(0.05 mmol, 10 mol %) in THF (1 mL) at room temperature
under N2 atmosphere. After the reaction was completed, the same procedure
was followed as for the sulfonyl amidines.
8. Morita, T.; Okamoto, Y.; Sakurai, H. Tetrahedron Lett. 1978, 28, 2523.
9. Those activities were evaluated in breast cancer cells by cell viability assay,
adipocytes derived 3T3-L1 cells by oil red S stained fat level, and MC3T3-E1
subclone 4 cells by measuring the deposited calcium level, respectively.
10. Boyle, W. J.; Simonet, W. S.; Lacey, D. L. Nature 2003, 423, 337.
11. NIH Consensus Development Panel on Osteoporosis Prevention, Diagnosis, and
Therapy. JAMA 2001, 285, 785.
12. Dannemann, C.; Gratz, K. W.; Riener, M. O.; Zwahlen, R. A. Bone 2007, 40,
828.
13. TRAP activity assay was performed as the following; mouse monocyte
RAW264.7 cells purchased from the American Type Culture Collection (VA,
References and notes
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USA) were suspended in
activator of nuclear factor-
a
j
-MEM with 10% FBS and 100 ng/ml of receptor
B ligand, and plated in 96-well plates at a density
of 1 Â 103 cells/well. After 24 h, amidine compounds (1 or 2) were treated into
cells. On differentiation day 4, cells were fixed with 10% formalin for 10 min
4. Kim, S. H.; Jung, D. Y.; Chang, S. J. Org. Chem. 2007, 72, 9769.
and 95% ethanol for 1 min, and then 100 ll of citrate buffer (50 mM, pH 4.6)
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6. General procedure for the preparation of sulfonyl amidines (1): To a stirred
mixture of alkyne (0.5 mmol, 1.0 equiv), azide (0.6 mmol, 1.2 equiv), and CuI
(0.05 mmol, 10 mol %) in THF (1 mL) was slowly added amine nucleophile
(0.6 mmol, 1.2 equiv) at room temperature under an N2 atmosphere. After the
reaction was completed, which was monitored with TLC, the reaction mixture
containing 10 mM of sodium tartrate and 5 mM of p-nitrophenylphosphate
(Sigma) was added to the fixed cells-containing wells. After incubation for 1 h,
the enzyme reaction mixtures in the wells were transferred into new plates
containing an equal volume of 0.1 N NaOH. Absorbance was measured at
410 nm with a Wallac EnVision HTS microplate reader (PerkinElmer, Finland).
The experiment was performed in triplicate.
14. No cytotoxicity of 1l or 2a was observed up to 10 lM (data not shown).