Synthesis and antiosteoclast activity of Di(1-oxo/thioxoperhydro- 1λ5-[1,3,2] diazaphospholo [1,5-a]pyridine-1-yl) (4-substituted phenyl) boronates

Article abstract of DOI:10.1002/hc.21010

A new family of di(1-oxo/thioxoper-hydro-1λ⁵-[1,3,2] diazaphospholo[1,5-a]pyridine-1-yl)(4-substituted phenyl) boronates (4a-j) has been synthesized in a two-step process. A reaction of (±)-piperidin-2-yl- methanamine (1) phosphoryl/phosphorothioyl chloride in the presence of triethylamine in dry tetrahydrofuran formed the intermediate monochloride (2), which on condensation with p-substituted phenylboronic acids (3a-j) afforded the titled compounds (4a-j). They were characterized by elemental, IR, ¹H, ¹³C, ³¹P NMR, and mass spectral analyses. All these compounds showed moderate to high antiosteoclast and osteoblast activity.

Full text of DOI:10.1002/hc.21010

Heteroatom Chemistry
Volume 23, Number 3, 2012
Synthesis and Antiosteoclast Activity of
5
Di(1-oxo/thioxoperhydro-1λ -[1,3,2]
diazaphospholo [1,5-a]pyridine-1-yl)
(4-substituted phenyl) Boronates
G. Chandra Sekhar Reddy,1 M. Veera Narayana Reddy,2 C. Radha
Rani,1 N. Bakthavatchala Reddy,1 K. Uma Maheswara Rao,1
S.K. Nayak,3 and C. Suresh Reddy1
1Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, India
2Department of Image Science and Engineering, Pukyong National University,
Busan 608 737, Korea
3Bio-Organic Division, Bhabha Atomic Research Centre, Mumbai 400 085, India
Received 14 July 2011; revised 22 December 2011
INTRODUCTION
ABSTRACT: A new family of di(1-oxo/thioxoper-
hydro-1λ5-[1,3,2]diazaphospholo[1,5-a]pyridine-1-yl)
Recent advances in the demands of industry,
(4-substituted phenyl) boronates (4a–j) has been
synthesized in a two-step process. A reaction of
( )-piperidin-2-yl-methanamine (1) phosphoryl/
phosphorothioyl chloride in the presence of triethy-
lamine in dry tetrahydrofuran formed the intermediate
monochloride (2), which on condensation with p-
substituted phenylboronic acids (3a–j) afforded the
titled compounds (4a–j). They were characterized by
chemical technology, and medicine have opened
new opportunities to utilize organoboronphospho-
rus compounds. 1,3,2 O, N, and P heterocycles have
attracted great interest due to their valuable phar-
macological effects and potential for synthetic appli-
cations [1]. They were recently reported to possess
matrix metalloproteinase inhibitory, anticancer [2],
pesticidal [3], and antimicrobial [4] activities.
The diphosphonates (DPs) have numerous med-
ical applications as drugs for the treatment of bone
diseases associated with excessive resorption and
have higher affinity for bone mineral [5] and abil-
ity for the prevention of the transition of amorphous
calcium phosphate into crystalline hydroxyapatite
[6]. They help in reduction of hydroxyapatite solu-
bility in vitro [7]. In a number of animal models, they
are shown to be effective in arresting bone loss and in
improving the biochemical properties of bone. This
led to their successful use as drugs for Paget’s disease
[8] and osteoporosis [9,10].
1
elemental, IR, H, 13C, 31P NMR, and mass spectral
analyses. All these compounds showed moderate to
ꢀ
C
high antiosteoclast and osteoblast activity.
2012
Wiley Periodicals, Inc. Heteroatom Chem 23:247–253,
2012; View this article online at wileyonlinelibrary.com.
DOI 10.1002/hc.21010
Correspondence to: C. Suresh Reddy; e-mail: csrsvu@gmail.com.
Contract grant sponsor: Board of Research in Nuclear Sciences,
Bhabha Atomic Research Centre, Mumbai, India.
The discovery of borane-modified phosphate
Contract grant number: 2010/37C/26/BRNS/1424.
2012 Wiley Periodicals, Inc.
c
ꢀ
analogues, boranophosphates, opened
a
new
247

248 Reddy et al.
O
O
O
P
O
O
P
C
H
O
O
P
S
O
O
O
H
O
P
O
H
H
H B
O
H
Phosphorothioate
Methylphosphate
Phosphate
Boranophosphate
FIGURE 1 Phosphate and its isolobal analogues.
chapter in phosphorus chemistry, especially in nu-
cleotide and nucleic acid chemistry. They have great
potential as therapeutic and diagnostic agents, as the
modification confers many unique properties [11–
16]. In the past two decades, evidence suggesting that
boron is also essential for healthy bones and joint
function has been mounting, possibly via effects
on the balance and absorption of calcium, magne-
sium, and phosphorus [17]. Recent developments in
boron chemistry and pharmacology provided signif-
icant progress in designing new organoboron com-
pounds with other heteroatoms, which exhibit an-
tiosteoporotic [18,19] property.
ering these properties, organoboronphosphates are
promising candidates for exploring their synthesis
and the study of new types of modified DP analogues.
The idea that compounds embedded with both
the bioactive structural features of boron and phos-
phonates may have good antiosteoporosis activity
prompted the authors to synthesize several new
class of boronodiphosphonates (BDPs) by a simple
route through the reaction of bioactive groups sub-
stituted phosphorochloridates with phenyl boronic
acid. Fortunately, these compounds have noticeable
antiosteoporosis properties.
A new class of boron-containing nucleoside bo-
ranophosphates (Fig. 1) was first reported by the
Shaw lab in the early 1990s [20,21]. These com-
pounds contain a borane moiety in place of one of
the nonbridging phosphate oxygens, resulting in a
hydrolytically stable analogue of the phosphodiester
linkage. This modification imparts unique charac-
teristics to boranophosphate nucleotides and nu-
cleic acids [13–16]. A boranophosphate is isosteric
to the neutral methylphosphonate group but, like the
phosphate and phosphorothioate groups, retains an
identical negative charge. Furthermore, the borane
group is isoelectronic with the oxygen occurring in
a phosphate and pseudoisoelectronic with the sul-
fur found in a phosphorothioate. Although parti-
tioning experiments have demonstrated that bora-
nophosphates possess high water solubility, they are
more lipophilic than phosphates. This could allow
for increased cellular uptake and targeted delivery
[22]. In addition, boranophosphates have increased
nuclease resistance without affecting activation of
RNase H cleavage of RNA in RNA:boranophosphate
hybrids [23,24]. Importantly, the stability of the bo-
ranophosphate allows its administration as a single-
stranded siRNA, which may allow for increased ease
of delivery (due to decreased charge and size) and de-
creased immunoactivation [25]. Considering these
properties, boranophosphates are promising candi-
dates for exploring new types of modified nucleotide,
polynucleotide analogues, and in drug synthesis. The
labile boron complexes have also readily formed,
remarkably stable and generally crystalline solids
with an active phosphorus intermediate. Consid-
RESULTS AND DISCUSSION
Chemistry
The synthesis of the di(1-oxo/thioxoperhydro-1λ5-[1,
3,2]diazaphospholo[1,5-a]pyridine-1-yl)(4-substitu-
ted phenyl) boronates (4a–j) has been accom-
plished through a two-stage process (Scheme 1).
The condensation of ( )-piperidin-2-yl-methana-
mine (1) with phosphoryl/phosphorothioyl chloride
in dry tetrahydrofuran (THF) in the presence of
SCHEME
1
Synthesis of di(1-oxo/thioxoperhydro-
1λ5-[1,3,2]diazaphospholo[1,5-a]pyridine-1-yl) (4-substituted
phenyl) boronates (4a–j).
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Antiosteoclast Activity of Title Diphosphonate Compounds 249
triethylamine (TEA) at 0–25◦C affords the corre-
recorded on a Bruker 400 and 500 MHz NMR spec-
trometer operating at 500 MHz for H, 125 MHz
1
sponding monochloride intermediate 1-chloroocta-
hydro-1λ5 -[1,3,2]diazaphospholo[1,5-a] pyridine-1-
oxide/thione (2). The reaction of 2 with various
substituted phenylboronic acids in dry THF in the
presence of TEA at 40–45◦C for 2–3 h with stirring
afforded the title compounds 4a–j in high yields.
All the compounds 4a–j exhibited infrared (IR)
spectrum absorption bands for P O, P S, and
P NH in the region of 1261–1268, 735–756, and
3296–3409 cm−1, respectively [26]. In the proton nu-
clear magnetic resonance (1H NMR) spectrum, all
the aromatic protons resonate as multiplets at δH
7.30–8.04 ppm. The NH proton gave a broad signal
at δH 2.68–2.86 ppm due to its coupling with phos-
phorus and neighboring methylene hydrogens [27].
The NH CH2 and N CH2 protons resonated as mul-
tiplets [27] at δH 2.90–3.14 ppm and δH 2.58–2.74
ppm, respectively. The N CH proton experienced
coupling with the adjacent two methylene protons
and resonated as a multiplet at δH 3.52–2.21 ppm.
All the remaining CH2 protons in the cyclohexane
ring also resonated as multiplets in the region of δH
1.10–1.86 ppm, due to this reason cyclohexane ring
that may exist in a chair form [28].
for 13C, and 202 MHz for 31P NMR. Spectra were
recorded in DMSO-d6 and referenced to TMS (1H and
13C) and 85% H3PO4 (31P). APCI-MS were recorded
on a Jeol SX102 DA/600 mass spectrometer. Elemen-
tal analyses were performed on a Thermo Finnigan
instrument at the University of Hyderabad, Hyder-
abad, India. The purity of the compounds was tested
on an HPLC-Shimadzu instrument using a C-18
column.
Structure–Activity Relationships
The two phosphonate groups have high affinity for
bone mineral with an electronegative atom or groups
together and act as a “bone hook,” allowing rapid
and efficient targeting of DPs to bone mineral sur-
faces. Once localized within bone, the structure and
three-dimensional conformation of the side chain as
well as substituents in the phosphonate groups in the
molecule determine the course of biological activity.
The most potent DPs contain 1 or 2 nitrogen
atoms in a heterocyclic moiety with alkyl side chain
attached to a DP group. The nitrogen-containing
diphosphonates are pharmaceutical agents possess-
ing a pyrophosphate-like chemical substructure that
confers a strong affinity for calcium. This tridentates
structure causes them to chelate circulating calcium
and to bind the mineral at bone surfaces.
In the carbon nuclear magnetic resonance (13C
NMR) spectra of 4a, 4b, 4e, 4h, and 4j, all the
aromatic carbons resonated at δC 120.7–160.8 ppm
and the C-9 resonated at δC 58.2–59.3 ppm. The re-
maining carbon chemical shifts were observed in
the expected region [27]. The phosphorus nuclear
magnetic resonance (31P NMR) chemical shifts of
P O and P S were resonated in the region of δP
16.72–18.15 ppm and 67.48–72.62 ppm [26], respec-
tively. The atmospheric pressure chemical ioniza-
tion mass spectra (APCI-MS) of 4a, 4b, 4e, 4h, and
4j showed peaks at m/z values corresponding to their
protonated molecular ions. In bioactivity, the antios-
teoporosis activity of compound 4d exhibited very
high osteoblast property and 4b, 4e, 4g, and 4i also
showed significant osteoblast activity.
Biological Activity
The effect on bone resorption of the new compounds
was investigated in terms of the structure–activity
relationships of DPs. Postmenopausal osteoporo-
sis is observed with two important cellular mech-
anisms, that is, with reduced osteoblast survival as
well as increased osteoclast functions. Hence, for in-
hibiting osteoclast functions or inducing osteoclast
cell death, ensuring osteoblast survival becomes es-
sential. Therefore, all the synthesized BDPs (4a–
j) were first tested in primary osteoblast cells for
their effect on cell viability using the MTT (3-(4,5-
dimethyl thiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay [29] at concentrations ranging from 10
pM to 1 μM. The screening results showed that the
most active BDPs were found in the compounds
4d and 4g that involve the bromo and fluoro sub-
stituents, respectively, and they show a closer activ-
ity to that of the standard compound. The moder-
ate activity appeared with compounds 4b, 4e, and
4i, whereas 4c and 4h had less effect on osteoblast
viability and they contain the chloro substituents
(Tables 1 and 2). Alendronate (Aln) was used as the
standard, and 100 nM of Aln maximally inhibited
EXPERIMENTAL
Chemicals procured from Sigma–Aldrich (Hyder-
abad, India), Merck (Mumbai, India), and Lancaster
Chemical (Mumbai, India) were used as such with-
out further purification. All solvents used for spec-
troscopic and other physical studies were reagent
grade and were further purified, employing the re-
ported methods. Melting points were determined us-
ing a calibrated thermometer by Guna Digital melt-
ing point apparatus. IR spectra were recorded on
a Perkin–Elmer FT-IR 240-C spectrophotometer us-
1
ing KBr optics. H, 13C, and 31P NMR spectra were
Heteroatom Chemistry DOI 10.1002/hc

250 Reddy et al.
TABLE
1
Compounds and Their Osteoclast Inhibitory
Osteoclast Inhibitory Ratio Mean SDa
(10 mL) and TEA (0.002 mol), the intermediate
monochloride 2 solution was added dropwise at 0◦C.
After the addition, the temperature was slowly raised
to 40–45◦C and the solution was stirred for 2 h. The
completion of the reaction was ascertained by TLC
conducted on a 3:7 mixture of ethylacetate and hex-
ane with an average Rf 0.70. The reaction mixture
was filtered to remove TEA hydrochloride, and the
filtrate on evaporation in a rotaevaporator afforded
the crude product. It was further purified by col-
umn chromatography on 60–120 mesh silica gel with
ethyl acetate:hexane (1:4) as an eluent. The crude
product was further purified by crystallization, and
the title compounds (4a–j) were characterized by IR,
1H, 13C, 31P NMR, and mass spectral data.
Activity
Compounds
4a
4b
4c
4d
4e
4f
36.1 6.5
61.4 2.5
28.4 3.1
68.7 3.1
60.6 5.6
22.1 1.7
66.4 4.3
19.3 4.2
62.7 2.6
48.8 3.4
70.7 6.3
4g
4h
4i
4j
Alnb
aThe inhibitory ratio (%) of BDPs’ μ of 4a–j were analyzed by MTT
assay after 8 days.
bAlendronate (standard reference).
Spectral and Analytical Data of Newly
Synthesized Compounds
TABLE 2 Effects of Borondiphosphonates on Rat Calvaria
Osteoblast Proliferation at Different Concentrations
Di(1-thioxoperhydro-1λ5 -[1,3,2]diazaphospholo
Compound
10−5M
10−6M
10−7M
10−8M
[1,5-a]pyridine-1-yl)phenylboronate
(4a). Yield,
82%; mp: 122–124◦C; IR (KBr) υmax [cm−1]: 3395
( NH), 748 (P S); 1H NMR (DMSO-d6) δ (ppm):
7.73 (2H, dd, Ar Hortho), 7.32 (2H, m, Ar Hpara),
7.36 (1H, m, Ar Hmeta), 3.41–3.22 (2H, m, N CH),
3.01–2.91 (4H, m, NH CH2), 2.70–2.62 (4H, m,
N CH2), 2.83–2.72 (2H, br, NH), 1.83–1.45 (8H,
m, 2×CH2), 1.30–1.14 (4H, m, CH2); 13C NMR
δ (ppm): 157.7 (C-1ꢁ), 134.1 (C-2ꢁ and C-6ꢁ), 130.3
(C-4ꢁ), 128.7 (C-3ꢁ and C-5ꢁ), 58.2 (C-9), 51.5 (C-3),
45.3 (C-7), 39.9 (C-4), 39.6 (C-6), 39.0 (C-5); 31P
NMR δ (ppm): 68.81; APCI-MS m/e: 470 (M+), 469,
378, 192 (100%). Anal. Calcd for C18H29BN4O2P2S2:
C, 45.97; H, 6.21; N, 11.91. Found: C, 45.88; H, 6.19;
N, 11.85.
4a
4b
4c
4d
4h
26.5 2.4 35.2 3.7 56.8 5.1 20.2 2.1
21.3 2.2 28.4 2.8 38.3 3.7 25.8 2.5
17.1 1.9 20.3 2.1 23.7 2.2 25.3 2.7
32.4 2.9 39.8 3.3 67.4 7.6 40.6 3.4
28.2 2.8 32.6 3.8 38.5 4.1 27.7 2.7
osteoclastogenesis, which was determined by the
number of TRAP (tartarate resistant acid phos-
phatase) positive cells. The biphasic antiosteoclastic
action of Aln has been reported in multiple myeloma
bone disease [30]. Therefore, the effects of various
BDPs were compared at the 100-nM concentration
along with Aln. Thus, the results obtained are en-
couraging for further optimization of the antiresorp-
tive properties of these compounds and could be
used to treat humans in the future.
Di(1-thioxoperhydro-1λ5 -[1,3,2]diazaphospholo
[1,5-a]pyridine-1-yl)(4-fluorophenyl) Boronate (4b).
Yield, 80%; mp: 105–107◦C; IR (KBr) υmax (cm−1):
3409 ( NH), 756 (P S); 1H NMR (DMSO-d6) δ
(ppm): 7.93 (2H, dd, Ar Hortho), 7.79 (2H, m,
Ar Hmeta), 3.50–3.26 (2H, m, N CH), 3.13–2.92 (4H,
m, NH CH2), 2.72–2.64 (4H, m, N CH2), 2.86–2.74
(2H, br, NH), 1.81–1.40 (8H, m, 2 × CH2), 1.28–1.11
(4H, m, CH2); 13C NMR δ (ppm): 160.8 (C-1ꢁ), 136.1
(C-2ꢁ and C-6ꢁ), 147.2 (C-4ꢁ), 126.0 (C-3ꢁ and C-5ꢁ),
59.2 (C-9), 56.8 (C-3), 40.1 (C-7), 40.0 (C-4), 39.8
(C-6), 39.3 (C-5); 31P NMR δ (ppm): 67.48; APCI-
MS m/e: 488 (M+), 484, 483 (100%). Anal. Calcd
for C18H28BFN4O2P2S2: C, 44.27; H, 5.78; N, 11.47.
Found: C, 44.14; H, 5.66; N, 11.39.
General Procedure for the Preparation of
Di(1-oxo/thioxoperhydro-1λ5-[1,3,2]
diazaphospholo[1,5-a]pyridine-1-yl)(4-
substituted phenyl) Boronates (4a–j)
A solution of phosphoryl/phosphorothioyl chloride
(0.002 mol) in dry THF (10 mL) was added dropwise
over a period of 15 min to a stirred solution of ( )-
piperidin-2-yl-methanamine (1, 0.002 mol) and TEA
(0.004 mol) in THF (10 mL) at 0–20◦C. After stirring
for 1 h at room temperature, formation of intermedi-
ate monochloride 2 was ascertained by TLC analysis,
TEA hydrochloride was removed from the reaction
mixture by filtration. This solution of intermediate
is used in the next stage without purification.
Di(1-thioxoperhydro-1λ5 -[1,3,2]diazaphospholo
[1,5-a]pyridine-1-yl)(4-chlorophenyl) Boronate (4c).
Yield, 78%; mp: 125–127◦C; IR (KBr) υmax (cm−1):
To a stirred solution of various p-substituted
phenylboronic acid (3a–j, 0.001 mol) in dry THF
Heteroatom Chemistry DOI 10.1002/hc

Synthesis and Antiosteoclast Activity of Title Diphosphonate Compounds 251
3401 ( NH), 742 (P S); 1H NMR (DMSO-d6)
3345 ( NH), 1261 (P O); 1H NMR (DMSO-d6)
δ (ppm): 7.93 (2H, dd, Ar Hortho), 7.81 (2H, m,
Ar Hmeta), 3.52–3.28 (2H, m, N CH), 3.04–2.91 (4H,
m, NH CH2), 2.72–2.65 (4H, m, N CH2), 2.86–2.74
(2H, br, NH), 1.80–1.40 (8H, m, 2×CH2), 1.30–1.14
(4H, m, CH2); 31P NMR δ (ppm): 17.79. Anal. Calcd
for C18H28BFN4O4P2: C, 47.39; H, 6.19; N, 12.28.
Found: C, 47.27; H, 6.08; N, 12.21.
δ (ppm): 7.75 (2H, dd, Ar Hortho), 7.49 (2H, m,
Ar Hmeta), 3.46–3.23 (2H, m, N CH), 3.04–2.90 (4H,
m, NH CH2), 2.70–2.61 (4H, m, N CH2), 2.86–2.72
(2H, br, NH), 1.80–1.40 (8H, m, 2×CH2), 1.30–1.10
(4H, m, CH2); 31P NMR δ (ppm): 72.62. Anal. Calcd
for C18H28BClN4O2P2S2: C, 42.83; H, 5.59; N, 11.10.
Found: C, 42.80; H; 5.51; N, 11.04.
Di(1-thioxoperhydro-1λ5 -[1,3,2]diazaphospholo
[1,5-a]pyridine-1-yl)(4-bromophenyl) Boronate (4d).
Yield, 83%; mp: 128–130◦C; IR (KBr) υmax (cm−1):
3392 ( NH), 752 (P S); 1H NMR (DMSO-d6) δ
(ppm): 7.61 (2H, dd, Ar Hortho), 7.43 (2H, m,
Ar Hmeta), 3.45–3.22 (2H, m, N CH), 3.03–2.90 (4H,
m, NH CH2), 2.70–2.61 (4H, m, N CH2), 2.85–2.71
(2H, br, NH), 1.80–1.40 (8H, m, 2×CH2), 1.25–1.10
(4H, m, CH2); 13C NMR δ (ppm): 160.1 (C-1ꢁ), 135.3
(C-2ꢁ and C-6ꢁ), 138.4 (C-4ꢁ), 120.7 (C-3ꢁ and C-5ꢁ),
59.0 (C-9), 56.3 (C-3), 40.1 (C-7), 39.9 (C-4), 39.5 (C-
6), 39.1 (C-5); 31P NMR δ (ppm): 68.21. Anal. Calcd
for C18H28BBrN4O2P2S2: C, 39.36; H, 5.14; N, 10.20.
Found: C, 39.25; H, 5.03, N; 10.11.
Di(1 - oxoperhydro - 1λ5 - [1,3,2]diazaphospholo
[1, 5-a]pyridine-1-yl)(4-chlorophenyl) Boronate (4h).
Yield, 82%; mp: 95–97◦C; IR (KBr) υmax (cm−1):
3393 ( NH), 1268 (P O); 1H NMR (DMSO-d6)
δ (ppm): 7.79 (2H, dd, Ar Hortho), 7.51 (2H, m,
Ar Hmeta), 3.48–3.23 (2H, m, N CH), 3.02–2.90 (4H,
m, NH CH2), 2.70–2.62 (4H, m, N CH2), 2.85–2.73
(2H, br, NH), 1.81–1.42 (8H, m, 2×CH2), 1.28–1.14
(4H, m, CH2); 31P NMR δ (ppm):18.15; APCI-MS
m/e: 474 (M + 2), 472 (M+), 445, 444 (100%),
417, 415, 361, 202, 200, 175, 159. Anal. Calcd for
C18H28BClN4O4P2: C, 45.74; H, 5.97; N, 11.85. Found:
C, 45.66; H, 5.91; N, 11.80.
Di(1 - oxoperhydro - 1λ5 - [1,3,2]diazaphospholo
[1, 5-a]pyridine-1-yl)(4-bromophenyl) Boronate (4i).
Yield, 79%; mp: 108–110◦C; IR (KBr) υmax (cm−1):
3375 ( NH), 1263 (P O); 1H NMR (DMSO-d6)
δ (ppm): 7.66 (2H, dd, Ar Hortho), 7.45 (2H, m,
Ar Hmeta), 3.45–3.20 (2H, m, N CH), 3.03–2.90 (4H,
m, NH CH2), 2.71–2.61 (4H, m, N CH2), 2.85–2.72
(2H, br, NH), 1.81–1.41 (8H, m, 2×CH2), 1.23–1.10
(4H, m, CH2); 31P NMR δ (ppm): 16.81. Anal. Calcd
for C18H28BBrN4O4P2: C, 41.81; H, 5.46; N, 10.83.
Found: C, 41.70; H, 5.35; N, 10.75.
Di(1-thioxoperhydro-1λ5 -[1,3,2]diazaphospholo
[1,5-a]pyridine-1-yl)(4-nitrophenyl) Boronate (4e).
Yield, 85%; mp: 88–90◦C; IR (KBr) υmax (cm−1): 3408
( NH), 735 (P S); 1H NMR (DMSO-d6) δ (ppm): 8.03
(2H, dd, Ar Hortho), 7.84 (2H, m, Ar Hmeta), 3.50–
3.24 (2H, m, N CH), 3.12–2.91 (4H, m, NH CH2),
2.70–2.62 (4H, m, N CH2), 2.85–2.73 (2H, br, NH),
1.81–1.41 (8H, m, 2×CH2), 1.30–1.12 (4H, m, CH2);
13C NMR δ (ppm): 160.7 (C-1ꢁ), 136.0 (C-2ꢁ and C-6ꢁ),
147.5 (C-4ꢁ), 126.2 (C-3ꢁ and C-5ꢁ), 59.0 (C-9), 56.5
(C-3), 40.1 (C-7), 40.0 (C-4), 39.5 (C-6), 39.3 (C-5);
31P NMR δ (ppm): 68.86; APCI-MS m/e: 515 (M+),
513, 341, 327 (100%), 247, 222, 197. Anal. Calcd for
C18H28BN5O4P2S2: C, 41.95; H, 5.48; N, 13.59. Found:
C, 41.84; H, 5.39; N, 13.52.
Di(1 - oxoperhydro - 1λ5 - [1,3,2]diazaphospholo
[1, 5-a]pyridine-1-yl)(4-nitrophenyl) Boronate (4j).
Yield, 76%; mp: 74–76◦C; IR (KBr) υmax [cm−1]:
3296 ( NH), 1267 (P O); 1H NMR (DMSO-d6)
δ (ppm): 8.04 (2H, dd, Ar Hortho), 7.88 (2H, m,
Ar Hmeta), 3.51–3.26 (2H, m, N CH), 3.14–2.92 (4H,
m, NH CH2), 2.72–2.63 (4H, m, N CH2), 2.85–2.74
(2H, br, NH), 1.81–1.42 (8H, m, 2×CH2), 1.30–1.16
(4H, m, CH2); 13C NMR δ (ppm): 160.8 (C-1ꢁ), 136.3
(C-2ꢁ and C-6ꢁ), 147.8 (C-4ꢁ), 126.1 (C-3ꢁ and C-5ꢁ),
59.3 (C-9), 56.7 (C-3), 40.2 (C-7), 39.9 (C-4), 39.7 (C-
6), 39.4 (C-5); 31P NMR δ (ppm): 19.48; APCI-MS
m/e: 483 (M+), 352, 256, 242 (100%), 182, 116. Anal.
Calcd for C18H28BN5O6P2: C, 44.74; H, 5.84; N, 14.49.
Found: C, 44.62; H, 5.75; N, 14.41.
Di(1 - oxoperhydro - 1λ5 - [1,3,2]diazaphospholo
[1, 5-a]pyridine-1-yl)phenylboronate (4f). Yield,
76%; mp: 132–134◦C; IR (KBr) υmax (cm−1): 3370
1
( NH), 1265 (P O); H NMR (DMSO-d6) δ (ppm):
7.76 (2H, dd, Ar Hortho), 7.36 (1H, m, Ar Hpara),
7.30 (2H, m, Ar Hmeta), 3.44–3.25 (2H, m, N-CH),
3.02–2.92 (4H, m, NH CH2), 2.70–2.62 (4H, m,
N CH2), 2.82–2.72 (2H, br, NH), 1.85–1.48 (8H, m,
2×CH2), 1.32–1.15 (4H, m, CH2); 31P NMR δ (ppm):
16.72. Anal. Calcd for C18H29BN4O4P2: C, 49.34; H,
6.67; N, 12.79. Found: C, 49.01; H, 6.53; N, 12.59.
Materials and Methods of Bioactivity Evaluation
Di(1 - oxoperhydro - 1λ5 - [1,3,2]diazaphospholo
[1, 5-a]pyridine-1-yl)(4-fluorophenyl) Boronate (4g).
Yield, 80%; mp 101–103◦C; IR (KBr) υmax (cm−1):
Osteoclasts were prepared from thighbone, shoul-
der bone, and tibia of 24 h newborn rat. The cells
Heteroatom Chemistry DOI 10.1002/hc

252 Reddy et al.
were stained with TRAP to count the number of
positive cells. Primary osteoblast-enriched cultures
were prepared from newborn Albino rat calvarias
at axenic circumstances. Parietal bones were dis-
sected free from the sutures. The periosteal layers
from both sides of the bones were removed care-
fully. The bones were cut into chips and digested
with pancreas for 30 min. After centrifugation, the
precipitated cells were diluted with Dulbecco’s mod-
ified Eagles medium. Then the obtained osteoblast-
enriched cell suspension was seeded in the culture
flask and cultured at 37◦C in the presence of 5% CO2.
The medium was changed every 3 days. Cell viability
was measured using a MTT cell proliferation assay.
Absorbance readings of the wells were taken at 492
nm. All the makers of osteoblastic differentiation val-
ues were normalized to the relative number of viable
cells as determined directly in the 96-well plates us-
ing the above-mentioned proliferation assay.
CONCLUSIONS
The present investigations describe an efficient and
simple approach to the synthesis of a variety of the
title DPs in satisfactory yields with the use of eas-
ily available starting materials. The pharmacological
evaluation discusses the antiresorptive activities of
the obtained products. Therefore, the study of these
results suggest that the compounds have beneficial
effects in osteoblast and this holds the hope of devel-
oping a new generation of antiresorption drugs after
further fine-tuning of their chemical structure and
detailed pharmacological studies.
ACKNOWLEDGMENTS
The authors thank Professor C. Devendranath
Reddy, Department of Chemistry, Sri Venkateswara
University, Tirupati, India, for his valuable advice.
The osteoclast precursor cells were plated at a
density 5 × 104 cells/well in a 96-well plate cultured
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