Anti-neoplastic activity of 1,3-diaza-2-functionalized-adamantan-6-one compounds against melanoma cells

Article abstract of DOI:10.2174/15734064113099900002

Four series of 1,3-diaza-2-functionalized-adamantan-6-one derivatives, bearing at the 2 position SO, SO2, POCl and PO2H functional groups, were synthesized via a key quadruple Mannich reaction, followed by transformation of an aminal functionality into th

Full text of DOI:10.2174/15734064113099900002

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Medicinal Chemistry, 2014, 10, 27-37
27
Anti-Neoplastic Activity of 1,3-Diaza-2-Functionalized-Adamantan-6-One
Compounds Against Melanoma Cells
Yifat Sharabi-Ronen, Shlomo Levinger, Miri Ben-Dahan Lellouche and Amnon Albeck*
The Julius Spokojny Bioorganic Chemistry Laboratory, Department of Chemistry, Bar-Ilan University, Ramat-Gan
52900, Israel
Abstract: Four series of 1,3-diaza-2-functionalized-adamantan-6-one derivatives, bearing at the 2 position SO, SO2,
POCl and PO2H functional groups, were synthesized via a key quadruple Mannich reaction, followed by transformation of
an aminal functionality into the final 2-thia- and 2-phospha compounds. The compounds were tested for cytotoxic activity
against the mouse B16-F10 melanoma cell line. Malignant melanoma is notorious for its high resistance to chemotherapy,
and new anti-melanoma drugs are urgently needed. The 2-thia compounds exhibited poor proliferation inhibition activity,
but the 2-phospha derivatives showed significant activity, with IC50 values of 10-60 ꢀM. The compounds induced cell
death by G2/M cell cycle arrest, which led to apoptosis, as determined by Annexin V-FITC/PI staining, mitochondrial
membrane potential changes assessed by the JC-1 reagent, caspases 3 and 7 activation, and morphological changes.
Keywords: Diaza-adamantane, melanoma, anticancer drugs, cytotoxicity, apoptosis, cell cycle.
INTRODUCTION
which already excludes 40% of the patients. Furthermore,
Zelboraf suffers poor response duration, and resistance
mechanisms have already been observed [21]. Yervoy, a
monoclonal antibody, is effective in only 25% of the pa-
tients. It may induce severe and even fatal immunological
adverse effects resulting from T cell activation and prolifera-
tion [22, 23]. Thus, the development of anti-cancer agents
for the treatment of melanoma, either alone or in combina-
tion with other drugs, is still of utmost importance.
Malignant melanoma is one of the most aggressive hu-
man cancers. Advanced melanoma may become metastatic,
and has a very poor prognosis [1, 2]. Melanoma exhibits
high resistance to chemotherapy and irradiation. Cancer cells
may acquire drug resistance by improved DNA repair, drug
clearance, and defects in the apoptotic pathways [3]. Unlike
other resistant cancer types, melanoma is usually character-
ized by lack of p53 mutations [4]. The failure of anti-cancer
therapy in melanoma is, at least in part, due to impaired
apoptosis: down regulation of apoptotic proteins such as
Apaf-1 [4-6], and over-expression of anti-apoptotic proteins
(Bcl-2) [5,7,8] and survival proteins (survivin and NF-ꢀB)
[9-11]. Melanocytes secrete melanin and protect neighboring
epidermal cells from DNA damage by UV light [12]. There-
fore, it is not surprising that melanoma is also resistant to
radiation therapy.
1,3-Diaza-adamantane derivatives exhibit antibacterial
[24], and psychotropic [25] activity. Other synthetic deriva-
tives are sodium channel blockers [26] and ꢀ-opiate and ꢁ-
receptor ligands [27]. Physiologically active natural alka-
loids with the 1,3-diaza-adamantane structure are also known
[28]. 1,3-Diaza-adamantan-6-one and 1,3-diaza-2-phospha-
adamantan-6-one derivatives were previously evaluated
against sarcoma, carcinoma and leukemia in vivo, though no
mechanism was suggested for the observed activity [29].
Most of these compounds showed very low toxicity in mice.
Some adamantane derivatives exhibited anti-melanoma ac-
tivity, by inducing apoptosis via G2/M cell cycle arrest [11].
These data indicate that adamantanes and their 1,3-diaza
analogs are potential candidates for the development of anti-
melanoma drugs. We previously synthesized a library of 3,7-
diaza-bicyclo[3.3.1]nonan-9-ones (bispidinone) derivatives
[30], which could serve as precursors for the synthesis of
1,3-diaza-adamantan-6-ones. Thus, in the present study we
report on the cyclization of the parent 3,7-diaza-
bicyclo[3.3.1]nonan-9-one compounds to produce four
libraries of 2-thia- and 2-phospha-diaza-adamantanes, and
on their in vitro anti-melanoma activity.
Although some small molecules were found to have ac-
tivity against melanoma in vitro [11], for several years only
one drug, Dacarbazine, was approved for the treatment of
metastatic melanoma, with poor response (10-20% of the
patients) and even lower rates of cure (about 5%) [13]. After
a decade-long stagnation, three other drugs (Zelboraf [14,
15], Yervoy [16,17] and Peginterferon alfa-2b [18]) have
been approved in 2011 for the treatment of late-stage mela-
noma. Their long-term effectiveness is still to be studied.
Nevertheless, some limitations in their application are al-
ready evident. Zelboraf is only active against melanoma cells
with the V600E and V600K mutations in B- Raf [19,20],
*Address correspondence to this author at the Department of Chemistry, Bar
Ilan University, Ramat Gan 52900, Israel; Tel: 972-3-5318862;
Fax: 972-3-7384053; E-mail: amnon.albeck@biu.ac.il
1875-6638/14 $58.00+.00
© 2014 Bentham Science Publishers

28 Medicinal Chemistry, 2014, Vol. 10, No. 1
Sharabi-Ronen et al.
MATERIALS AND METHODS
Ph), 3.88 (d, J = 12.0 Hz, 4H, CCH2N), 3.71 (d, J = 12.0 Hz,
4H, CCH2N); 13C NMR ꢀ 210.1, 138.3, 128.1, 127.5, 127.2,
61.3, 56.3; HRMS m/z for C19H20N2O (M+): calcd 292.1576,
found 292.1588.
Chemistry
General
1H NMR spectra were recorded at 200, 300 or 600 MHz,
13C NMR spectra at 50.3, 75.5 or 150.9 MHz, 31P NMR
spectra at 81 MHz, and 19F NMR spectra at 188.3 MHz, all
1,5-Dibenzyl-3,7-diaza-bicyclo[3.3.1]nonan-9-one (4d).
1
27% yield. H NMR ꢀ 7.28-7.09 (m, 10H, Ph), 3.30 (d, J =
12.0 Hz, 4H, CCH2N), 2.97 (d, J = 12.0 Hz, 4H, CCH2N),
2.81 (s, 4H, CH2Ph), 2.63 (bs, 2H, NH); 13C NMR ꢀ 214.3,
137.1, 130.6, 128.1, 126.4, 59.5, 52.2, 38.1; HRMS m/z for
C21H25N2O (MH+): calcd 321.1967, found 321.1968.
1
in CDCl3, unless otherwise specified. H and 13C chemical
shifts are reported in ppm relative to TMS in CDCl3 or rela-
tive to solvent resonance in other solvents; for other nuclei
an external reference was used. Multiplicities designated as
“d” and “t” refer to resonances with 2nd order character in
AA’XX’ systems. 13C Signal multiplicity was determined by
the DEPT technique. HMQC and HMBC methods were used
to establish atom connectivities. HRMS was recorded in
DCI+ mode with methane as reagent gas, unless otherwise
indicated. Chromatography refers to flash column chroma-
tography, carried out on silica gel 60 (230-400 mesh ASTM,
E. Merck). TLC was performed on E. Merck 0.2 mm perco-
lated silica gel F-254 plates. Compounds were detected by
UV light (254 nm) and/or by staining with vanillin spray
reagent (1 g vanillin and 1 mL H2SO4 in 100 mL EtOH).
General Procedure for the Synthesis of Cyclic Sulfurous
Diamides 5
To a chloroform solution (3 mL) of diamine 4 (0.13
mmol) and triethylamine (71 ꢀL, 0.51 mmol) was added a
solution of distilled SOCl2 (14 ꢀL, 0.19 mmol) in chloroform
(1 mL) at 0 oC under N2 atmosphere. After stirring at rt for 5
h, the mixture was extracted with water and chloroform
(3x30 mL). The combined organic layer was washed with
saturated NaCl solution. Drying over MgSO4 and concentra-
tion in vacuum afforded the clean product.
5,7-Dibutyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-6-
1
Ketones 1d-f, 1,3-diaza-adamantan-6-ones 2a-f and diAl-
loc-bispidinones 3a-f were prepared as previously described
[27].
one 2-oxide (5a). 92% yield. H NMR ꢀ 4.45 ("d", J = 14.4
Hz, 2H, CCH2N), 3.90 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.40
(“d", J = 14.4 Hz, 2H, CCH2N), 2.83 (“d", J = 14.4 Hz, 2H,
CCH2N), 1.44-1.22 (m, 12H, Me(CH2)3), 0.93 (t, J = 6.9 Hz,
3H, Me), 0.92 (t, J = 7.1 Hz, 3H, Me); 13C NMR ꢀ 209.8,
60.1, 51.35, 47.0, 45.0, 31.6, 30.1, 25.4, 24.6, 23.6, 23.5,
14.0.
General Procedure for the Synthesis of Diamines 4
Alloc-protected diamine 3 (1 mmol), Pd(PPh3)4 (0.02
mmol) and AcOH (2.4 mmol) were dissolved in CH3Cl (20
mL) under argon atmosphere in a Schlenk tube equipped
with a magnetic bar and fitted with a rubber septum,. The
pale yellow color of the solution was then characteristic of
the presence of the palladium (II) complex. Bu3SnH (ca. 1.1
mmol) was then rapidly added to the stirred solution. A
slightly exothermic reaction with vigorous gas evolution
immediately ensued. At the end of the reaction (a few hours)
the golden yellow color of the reaction mixture indicated the
presence of palladium (0) species in the medium. The solu-
tion was diluted with CHCl3 and extracted with aqueous
NaHCO3. The organic layer was acidified by H2SO4 (200
mL H2O : 2 mL concentrated H2SO4), the aqueous layer was
washed with CHCl3, then made basic with NaOH 10% solu-
tion, and extracted with CHCl3. The organic layer was dried
over MgSO4.
5,7-Dipentyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-6-
1
one 2-oxide (5b). 88% yield. H NMR ꢀ 4.45 ("d", J = 14.1
Hz, 2H, CCH2N), 3.90 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.40
(“d", J = 14.4 Hz, 2H, CCH2N), 2.83 (“d", J = 14.4 Hz, 2H,
CCH2N), 1.47-1.22 (m, 16H, Me(CH2)4), 0.90 (t, J = 6.8 Hz,
3H, Me), 0.89 (t, J = 6.8 Hz, 3H, Me); 13C NMR ꢀ 209.8,
60.1, 51.36, 47.0, 45.1, 32.7, 32.6, 31.81, 30.4, 22.9, 22.5,
22.1, 14.1; HRMS m/z for C17H30N2O2S (M+): calcd
326.2028, found 326.1993.
5,7-Diphenyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-
1
6-one 2-oxide (5c). 100% yield. H NMR ꢀ 7.42-7.19 (m,
10H, Ph), 4.95 ("d", J = 14.4 Hz, 2H, CCH2N) 4.42 (“d”, J =
14.4 Hz, 2H, CCH2N) 4.15 ("d", J = 14.4 Hz, 2H, CCH2N)
3.59 ("d", J = 14.4Hz, 2H, CCH2N); 13C NMR ꢀ 205.2,
136.5, 135.3, 128.7, 128.6, 128.1, 128.0, 127.0, 126.6, 60.7,
51.7, 50.5, 48.9; HRMS m/z for C19H18N2O2S (M+): calcd
338.1089, found 338.1085.
1,5-Dibutyl-3,7-diaza-bicyclo[3.3.1]nonan-9-one (4a).
66% yield. 1H NMR ꢀ 3.41 (d, J = 12.0 Hz, 4H, eq CCH2N),
2.92 (d, J = 12.0 Hz, 4H, ax CCH2N), 2.77 (bs, 2H, NH),
1.39-1.15 (m, 12H, Me(CH2)3), 0.90 (t, J = 7.1 Hz, 6H, Me);
13C NMR ꢀ 215.7, 59.8, 51.8, 31.7, 25.7, 23.7, 14.0; HRMS
m/z for C15H29N2O (MH+): calcd 253.2280, Found 253.2250.
5,7-Dibenzyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-
1
6-one 2-oxide (5d). 99% yield. H NMR ꢀ 7.35-7.12 (m,
10H, Ph), 4.38 ("d", J = 14.1 Hz, 2H, CCH2N), 3.81 (“d”, J =
14.1 Hz, 2H, CCH2N), 3.34 ("d", J = 14.4 Hz, 2H, CCH2N)
2.88 (s, 2H, CH2Ph), 2.80 (“d", J = 14.4 Hz, 2H, CCH2N),
2.79 (s, 2H, CH2Ph); 13C NMR ꢀ 208.8, 135.3, 135.1, 130.7,
130.5, 128.7, 128.6, 127.1, 127.0, 59.5, 51.2, 47.6, 45.4,
37.9, 36.3; HRMS m/z for C21H22N2O2S (M+): calcd
366.1402, found 366.1371.
1,5-Dipentyl-3,7-diaza-bicyclo[3.3.1]nonan-9-one (4b).
30% yield. 1H NMR ꢀ 3.43 (d, J = 12.0 Hz, 4H, eq CCH2N),
3.01 (bs, 2H, NH), 2.93 (d, J = 12.0 Hz, 4H, ax CCH2N),
1.33-1.19 (m, 16H, Me(CH2)4), 0.88 (t, J = 7.1 Hz, 6H, Me);
13C NMR ꢀ 215.3, 59.6, 51.7, 32.9, 31.9, 23.1, 22.6, 14.1;
HRMS m/z for C17H33N2O (MH+): calcd 281.2593, found
281.2597.
5,7-Bis[(4-(trifluoromethyl)phenyl)methyl]-1,3-diaza-2-
thia-tricyclo[3.3.1.13,7]decan-6-one 2-oxide (5e). To 2e (1.0
mmol) in pyridine : acetonitrile 1:1 (2 mL) was added a solu-
1,5-Diphenyl-3,7-diaza-bicyclo[3.3.1]nonan-9-one (4c).
1
81% yield. Mp: 259-260 °C H NMR ꢀ 7.38-7.26 (m, 10H,

Diaza-adamantanones as Anti-melanoma Compounds
Medicinal Chemistry, 2014, Vol. 10, No. 1 29
tion of distilled SOCl2 (90 ꢀL, 1.2 mmol) in acetonitrile (1
CCH2N), 3.22 (d, J = 13.7 Hz, 4H, CCH2N), 2.91 (s, 4H,
CH2Ar); 13C NMR ꢀ 205.9, 139.1, 131.0, 125.8, 125.7, 60.9,
45.8, 35.4; HRMS m/z for C23H21F6N2O3S (MH+): calcd
519.1177, found 519.1153.
o
mL) at 0 C. The solution was stirred over night at rt, fol-
lowed by 1 h under reflux. The mixture was cooled, and
poured into ice-water bath. The crude product was collected,
washed with cold water, and purified by chromatography
5,7-Bis[(3,5-bis(trifluoromethyl)phenyl)methyl]-1,3-
diaza-2-thia-tricyclo[3.3.1.13,7]decan-6-one 2,2-dioxide (6f).
Was prepared as 6e in 17% yield. 1H NMR ꢀ 7.77 (s, 2H, Ar
(H-4)), 7.72 (s, 4H, Ar (H-2)) 4.31 (d, J = 13.5 Hz, 4H,
CCH2N), 3.22 (d, J = 13.5 Hz, 4H, CCH2N), 2.96 (s, 4H,
1
(CHCl3 + 1% MeOH). Yield: 151.5 mg (30%). H NMR ꢀ
7.58 (d, J = 7.8 Hz, 2H, Ar, (H-3)), 7.55 (d, J = 7.8 Hz, 2H,
Ar, (H-3)), 7.36 (d, J = 8.1 Hz, 2H, Ar (H-2)), 7.31 (d, J =
8.1 Hz, 2H, Ar (H-2)), 4.39 (“d”, J = 14.1, Hz, 2H, CCH2N),
3.82 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.35 ("d", J = 14.4 Hz,
2H, CCH2N) 2.93 (s, 2H, CH2Ar), 2.84 (s, 2H, CH2Ar), 2.79
(“d", J = 14.4 Hz, 2H, CCH2N); 13C NMR ꢀ 208.1, 139.6,
3
CH2Ar); 13C NMR ꢀ 205.4, 138.3, 130.9, 121.6 (septet, JCF
= 3.7 Hz), 61.0, 46.0, 35.4; HRMS m/z for C25H19F12N2O3S
(MH+): calcd 655.0925, found 655.0927.
3
3
131.2, 131.0, 125.7 (q, JCF = 3.8 Hz), 125.5 (q, JCF = 3.7
Hz), 59.6, 51.2, 47.7, 45.6, 37.6, 36.2; HRMS m/z for
C23H21F6N2O2S (MH+): calcd 503.1228, found 503.1246.
General Procedure for the Synthesis of Phosphorodiamidic
Chlorides 7
To a chloroform solution (3 mL) of diamine 4 (0.13
mmol) and triethylamine (71 ꢀL, 0.51 mmol) was added a
solution of distilled POCl3 (14 ꢀL, 0.19 mmol) in chloroform
(1 mL) at 0 oC under N2 atmosphere. After stirring at rt for 5
h, the mixture was extracted with ether and chloroform and
the combined organic layer was washed with brine. Drying
over MgSO4 and concentration in vacuum afforded the clean
product.
General Procedure for the Synthesis of Cyclic Sulfamides 6
To diamine 4 (0.3 mmol) in CHCl3 (5 mL) was added
o
Et3N (1.06 mmol). The solution was cooled to 0 C, treated
o
with SO2Cl2 (0.31 mmol), and stirred for 1 h at 0 C, fol-
lowed by 4 h at rt. After evaporation of the solvent, the mix-
ture was diluted with CHCl3 (40 mL) and washed with 0.5M
H3PO4, saturated NaHCO3 solution and brine, dried over
MgSO4 and evaporated.
5,7-Dibutyl-2-chloro-1,3-diaza-2-phospha-
5,7-Dibutyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-6-
one 2,2-dioxide (6a). 66% yield. 1H NMR ꢀ 3.21 (d, J = 10.5
Hz, 4H, eq CCH2N), 3.00 (d, J = 10.2 Hz, 4H, ax CCH2N),
1.56-1.16 (m, 12H, Me(CH2)3), 0.90 (t, J = 7.1 Hz, 6H, Me);
13C NMR ꢀ 207.6, 71.7, 59.2, 28.0, 26.7, 23.5, 13.9; HRMS
m/z for C15H26N2O3S (M+): calcd 314.1664, found 314.1698.
1
tricyclo[3.3.1.13,7]decane-6-one 2-oxide (7a). 82% yield. H
NMR ꢀ 4.41 (“d”, J = 14.4, Hz, 2H, CCH2N), 4.04 (“d”, J =
14.4 Hz, 2H, CCH2N), 3.27 (d“d", J = 32.7, 14.4 Hz, 2H,
CCH2N), 3.22 (d“d", J = 26.7, 14.4 Hz, 2H, CCH2N), 1.33
(m, 12H, Me(CH2)3), 0.93 (t, J = 6.6 Hz, 3H, Me), 0.91 (t, J
= 6.8 Hz, 3H, Me); 13C NMR ꢀ 208.3, 61.3 (d, J = 2.6 Hz),
60.3 (d, J = 2.6 Hz), 49.2 (d, J = 6.5 Hz), 47.0 (d, J = 6.5
Hz), 29.9, 29.8, 25.5, 25.4, 23.6, 14.0, 13.9; HRMS m/z for
C15H26ClN2O2P (M+): calcd 333.1499, found 333.1502.
5,7-Dipentyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-6-
one 2,2-dioxide (6b). 59% yield. 1H NMR ꢀ 3.22 (d, J = 11.1
Hz, 4H, eq CCH2N), 3.01 (d, J = 11.1 Hz, 4H, ax CCH2N),
1.69-1.26 (m, 16H, Me(CH2)4), 0.88 (t, J = 6.8 Hz, 6H, Me);
13C NMR ꢀ 71.9, 59.2, 32.7, 27.1, 25.6, 22.5, 14.1; HRMS
m/z for C17H31N2O3S (MH+): calcd 343.2055, found
343.2078.
2-Chloro-5,7-dipentyl-1,3-diaza-2-phospha-
1
tricyclo[3.3.1.13,7]decane-6-one 2-oxide (7b). 89% yield. H
NMR ꢀ 4.41 (“d”, J = 14.4 Hz, 2H, CCH2N), 4.03 (“d”, J =
14.4 Hz, 2H, CCH2N), 3.27 (d“d", J = 33.0, 14.4 Hz, 2H,
CCH2N), 3.21 (d"d", J = 26.7, 14.4 Hz, 2H, CCH2N), 1.44-
1.21 (m, 16H, Me(CH2)4), 0.90 (t, J = 6.5 Hz, 3H, Me), 0.88
(t, J = 6.9 Hz, 3H, Me); 13C NMR ꢀ 208.3, 61.3 (d, J = 2.7
Hz), 60.3 (d, J = 2.7 Hz), 49.3, 47.1, 32.6, 32.5, 30.1, 23.0,
22.97, 22.9, 22.5, 14.1.
5,7-Diphenyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-
1
6-one 2,2-dioxide (6c). 85% yield. H NMR ꢀ 7.38-7.07 (m,
10H, Ph), 3.67 (s, 8H, CCH2N); 13C NMR ꢀ 203.3, 133.9,
128.6, 128.4, 128.1, 71.6, 64.8; HRMS m/z for C19H18N2O3S
(M+): calcd 354.1038, found 354.0998.
5,7-Dibenzyl-1,3-diaza-2-thia-tricyclo[3.3.1.13,7]decan-
2-Chloro-5,7-diphenyl-1,3-diaza-2-phospha-
1
6-one 2,2-dioxide (6d). 64% yield. H NMR ꢀ 7.31-7.09 (m,
tricyclo[3.3.1.13,7]decane-6-one 2-oxide (7c). 100% yield.
1H NMR ꢀ 7.38-7.15 (m, 10H, Ph), 4.86 (“d”, J = 14.4 Hz,
2H, CCH2N) 4.48 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.95
(d"d", J = 27.0, 14.4 Hz, 4H, CCH2N); 13C NMR ꢀ 203.8,
134.7, 134.6, 128.5, 128.4, 127.9, 127.8, 127.0, 126.7, 61.5
(d, J = 2.3 Hz), 60.5 (d, J = 2.3 Hz), 52.4 (d, J = 5.3 Hz),
50.4 (d, J = 5.3 Hz); 31P-NMR ꢀ 35.91 (quintet, J = 27 Hz);
HRMS m/z for C19H19ClN2O2P (MH+): calcd 373.0873,
found 373.0873.
10H, Ph), 3.15 (d, J = 10.8 Hz, 4H, CCH2N), 2.87 (d, J =
10.8 Hz, 4H, CCH2N), 3.01(s, 4H, CH2Ph); 13C NMR ꢀ
206.6, 137.1, 130.1, 128.4, 126.6, 71.1, 59.3, 32.6; HRMS
m/z for C21H22N2O3S (M+): calcd 382.1351, found 382.1315.
5,7-Bis[(4-(trifluoromethyl)phenyl)methyl]-1,3-diaza-2-
thia-tricyclo[3.3.1.13,7]decan-6-one 2,2-dioxide (6e). To 1,3-
diaza-adamantan-6-one 2e (1.0 mmol) in pyridine : acetoni-
trile 1:1 (2 mL) was added a solution of distilled SO2Cl2 (100
o
ꢀL, 1.2 mmol) in acetonitrile (1 mL) at 0 C. The solution
5,7-Dibenzyl-2-chloro-1,3-diaza-2-phospha-
1
was stirred overnight at rt, followed by 1 h under reflux. The
mixture was cooled, and poured into ice-water bath. The
crude was collected and washed with cold water. Purification
by chromatography (CHCl3 + 1% MeOH), afforded 45 mg
tricyclo[3.3.1.13,7]decane-6-one 2-oxide (7d). 88% yield. H
NMR ꢀ 7.34-7.12 (m, 10H, Ph), 4.36 (“d”, J = 14.4 Hz, 2H,
CCH2N), 3.96 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.20 (d”d", J
= 32.7, 14.4 Hz, 2H, CCH2N), 3.16 (d"d", J = 26.7, 14.4 Hz,
1
(17% yield). H NMR ꢀ 7.56 (d, J = 8.1 Hz, 4H, Ar (H-3)),
2H, CCH2N), 2.88 (s, 2H, CH2Ph), 2.83 (s, 2H, CH2Ph); 13
C
7.32 (d, J = 8.1 Hz, 4H, Ar (H-2)), 4.25 (d, J = 13.7 Hz, 4H,
NMR ꢀ 207.4, 135.2, 130.6, 130.4, 128.7, 127.2, 127.1, 60.7

30 Medicinal Chemistry, 2014, Vol. 10, No. 1
Sharabi-Ronen et al.
(d, J = 2.5 Hz), 60.2 (d, J = 2.5 Hz), 49.7 (d, J = 6.1 Hz),
47.4 (d, J = 6.7 Hz), 36.1, 35.8; 31P NMR ꢀ 35.86 (quintet, J
= 29.4 Hz); HRMS m/z for C21H23ClN2O2P (MH+): calcd
401.1186, found 401.1233.
Sodium
5,7-bis[(4-(trifluoromethyl)phenyl)methyl]-6-
oxo-1,3-diaza-2-phospha-tricyclo[3.3.1.13,7]decan-2-olate 2-
1
oxide (8e). Was prepared as 8a in 100% yield. H NMR
(DMSO-d6) ꢀ 7.62 (d, J = 8.1 Hz, 4H, Ar (H-3)), 7.41 (d, J =
7.8 Hz, 4H, Ar (H-2)), 3.84 (d, J = 12.3 Hz, 4H, CCH2N),
2.75 (s, 4H, CH2Ar), 2.74 (dd, J = 19.8, 13.2 Hz, 4 H,
CCH2N); 13C NMR ꢀ 142.7, 131.3, 124.7, 60.6, 48.7, 36.0;
5,7-Bis[(4-(trifluoromethyl)phenyl)methyl]-2-chloro-1,3-
diaza-2-phospha-tricyclo[3.3.1.13,7]decane-6-one 2-oxide
(7e). To 1,3-diaza-adamantan-6-one 2e (1.0 mmol) in pyri-
dine : acetonitrile 1:1 (2 mL) was added a solution of dis-
tilled POCl3 (114 ꢀL, 1.2 mmol) in acetonitrile (1 mL) at 0
oC. The solution was stirred over night at rt, followed by 1h
under reflux. The mixture was cooled, and poured into ice-
water bath. The crude was collected and washed with cold
water. The product was purified by chromatography (CHCl3
31P NMR
ꢀ
2.19; HRMS (MALDI-TOF) m/z for
C23H20F6N2O3P (M-): calcd 517.1110, found 517.1140.
Pharmacology
Cell Culture
B16-F10 melanoma cell line was obtained from the
American Type Culture Collection (ATCC). The B16-F10
melanoma cells were cultured in RPMI 1640 medium (Bio-
logical Industries, Inc., Kibbutz Beit Haemek, Israel), sup-
plemented with 10% fetal calf serum (FCS), 1% penicillin–
streptomycin–nystatin, and 0.2% amphotericin. The cells
1
+ 1% MeOH). 33% yield. H NMR ꢀ 7.57 ("t", J = 8.4 Hz,
4H, Ar), 7.34 ("t", J = 8.4 Hz, 4H, Ar), 4.38 (“d”, J = 14.4,
Hz, 2H, CCH2N), 3.97 (“d”, J = 14.4 Hz, 2H, CCH2N), 3.19
(d”d", J = 32.7, 14.7 Hz, 4H, CCH2N) 2.93 (s, 2H, CH2Ar),
2.88 (s, 2H, CH2Ar); 13C NMR ꢀ 206.7, 139.5, 139.4, 131.1,
2
3
130.9, 129.5 (q, JCF = 42.2 Hz), 125.6 (q, JCF = 3.3 Hz),
60.7 (d, J = 2.3 Hz), 60.1 (d, J = 2.1 Hz), 49.7 (d, J = 6.2
Hz), 47.5 (d, J = 6.5 Hz), 35.9, 35.7; 31P NMR ꢀ 35.44;
HRMS m/z for C23H20ClF6N2O2P (M+): calcd 536.0855,
found 536.0848.
o
were maintained at 37 C and 5% CO2 in a humid environ-
ment.
Cell Proliferation and Viability Assay
The effect of the various 1,3-diaza-adamantan-6-one
compounds on cell proliferation was measured by a modified
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bro-
mide (MTT) assay. B16-F10 cells (5 x 103) were grown in
RPMI-10% FCS on a 96-well microtiter plate and the 1,3-
diaza-adamantan-6-one compounds (10-1000 mM in
DMSO) were added to a final concentration of 10-1000 ꢀM.
DMSO (at 0.1% concentration) was also added to control
wells, to account for the effect of the vehicle on the cells.
After 24 h of incubation, the medium was replaced with 130
ꢀl of fresh RPMI-1640 complete media and 20 ꢀl of MTT
reagent (5 mg/1 mL PBS) was added to each well. The cells
5,7-Bis[(3,5-bis(trifluoromethyl)phenyl)methyl]-2-
chloro-1,3-diaza-2-phospha-tricyclo[3.3.1.13,7]decane-6-one
1
2-oxide (7f). Was prepared as 7e in 31% yield. H NMR ꢀ
7.76 (m, 6H, Ar), 4.45 (“d”, J = 14.4, Hz, 2H, CCH2N), 4.01
(“d”, J = 14.4 Hz, 2H, CCH2N), 3.20 (d”d", J = 32.4, 14.7
Hz, 4H, CCH2N) 2.98 (s, 2H, CH2Ar), 2.95 (s, 2H, CH2Ar);
2
13C NMR ꢀ 206.2, 137.8, 137.6, 132.1 (q, JCF = 33.2 Hz),
132.0 (q, 2JCF = 32.9 Hz), 131.0, 130.8,125.0, 121.4, 60.9 (d.
J = 1.8 Hz), 60.1 (d, J = 1.8 Hz), 49.8 (d, J = 5.9 Hz), 47.8
(d, J = 5.9 Hz), 35.9, 35.8; 31P NMR ꢀ 35.27; HRMS m/z for
C25H18ClF12N2O2P (M+) calcd 673.0681, found 673.0645.
o
were incubated at 37 C for an additional 2 h. Cells were
Sodium 5,7-dibutyl-6-oxo-1,3-diaza-2-phospha-tricyclo
[3.3.1.13,7]decan-2-olate 2-oxide (8a). To 7a (0.3 mmol) in
THF (2 mL) was added a solution of 2M NaOH (700 ꢀL).
The solution was stirred over night at reflux, and then con-
centrated under reduced pressure to give the pure product in
subsequently lysed by the addition of 100 ꢀl DMF solution
(50% final concentration of DMF and 20% SDS, pH 4.7) to
each well, and incubated for 7 h. Absorption at 570 nm for
each well was measured using an ELISA reader. IC50 values
were calculated from cell viability data, and defined as the
approximate concentration resulting in 50% inhibition of cell
survival as compared to untreated cells.
1
100% yield. H NMR (DMSO-d6) ꢀ 3.89 (d, J = 13.0 Hz,
4H, eq CCH2N), 2.69 (dd, J = 21.0, 12.4 Hz, 4H, ax
CCH2N), 1.21 (m, 12H, Me(CH2)3), 0.81 (t, J = 6.5 Hz, 6H,
Me); 13C NMR ꢀ 211.9, 61.1, 47.4 (d, J = 5.0 Hz), 30.2,
25.2, 23.3, 13.9; 31P NMR ꢀ 3.39.
Annexin V-FITC/PI Flow Cytometry Staining Technique
Apoptosis was determined with an Annexin V-FITC kit
purchased from MBL Co. Ltd., according to the manufac-
turer’s instructions. B16-F10 cells (6 x 105) were seeded in
100 mm culture dishes, and allowed to attach overnight. The
cells were treated with 8e (50 ꢀM, 60 ꢀM, 75 ꢀM) and 7e
(25 ꢀM, 30 ꢀM) for 24 h, and then were collected and
washed with ice-cold PBS. To detect early and late apopto-
sis, both adherent and floating cells were harvested together.
The washed cell pellet was resuspended in ice-cold binding
buffer containing FITC-conjugated annexin V and PI. The
sample was incubated for 15 min in the dark before analysis
by flow cytometer.
Sodium 6-oxo-5,7-diphenyl-1,3-diaza-2-phospha-tricyclo
[3.3.1.13,7]decan-2-olate 2-oxide (8c). Was prepared as 8a in
1
86% yield. H NMR (DMSO-d6) ꢀ 7.38 (m, 10H, Ph), 4.42
(d, J = 12.8 Hz, 4H, CCH2N), 3.95 (dd, J = 22.2, 12.8 Hz,
4H, CCH2N); 13C NMR ꢀ 135.9, 128.6, 127.8, 126.9, 60.7,
51.8(d, J = 4.5 Hz); 31P NMR ꢀ 2.47; HRMS (MALDI-TOF)
m/z for C19H18N2O3P (M-): calcd 353.1050, found 353.107.
Sodium 5,7-dibenzyl-6-oxo-1,3-diaza-2-phospha-tricyclo
[3.3.1.13,7]decan-2-olate 2-oxide (8d). Was prepared as 8a in
100% yield. 1H NMR (DMSO-d6) ꢀ 7.19 (m, 10H, Ph), 3.84
(d, J = 12.8 Hz, 4H, eq CCH2N), 2.71 (dd, J = 21.0, 12.4 Hz,
4H, ax CCH2N), 2.65 (s, 4H, CH2Ph); 13C NMR ꢀ 211.2,
137.4, 130.5, 127.9, 126.0, 60.6, 48.5 (d, J = 5.0 Hz), 36.0;
31P NMR ꢀ 2.48 (quintet, J = 21 Hz).
JC-1 Mitochondrial Membrane Potential Detection Assay
The fluorescent cationic dye 5,5',6,6'-tetrachloro-
1,1',3,3'- tetraethyl-benzimidazolylcarbocyanine iodide (JC-

Diaza-adamantanones as Anti-melanoma Compounds
Medicinal Chemistry, 2014, Vol. 10, No. 1 31
1) was used for in situ detection of mitochondrial membrane
transition events in live cells, to provide an early indication
of the initiation of cellular apoptosis. JC-1 is internalized as
a monomer in the cytosol (green fluorescence, emission
wavelength 530 nm) and also accumulates as aggregates in
mitochondria with negative inner membrane potential (red
fluorescence, emission wavelength 590 nm). Consequently,
mitochondrial depolarization is indicated by a decrease in the
red/green fluorescence intensity ratio. For this assay, cells
were treated with 7e (20 - 40 ꢀM) and 8e (50 - 100 ꢀM) and
3,7-diaza-bicyclo[3.3.1]nonan-9-one derivatives 3a-f, typi-
cally in over 90% yield, and then were deprotected [33] to
yield 4a-f. These diamine compounds were then cyclized to
a library of diaza-adamantane derivatives bearing six 5,7-
dialkyl and diaryl substituents and three functional groups at
the 2 position, SO (5a-f) [34], SO2 (6a-f) [35,36], and POCl
(7a-f) [37]. The latter was hydrolyzed to yield the fourth
-
functional group PO2 (8a-f) (Scheme 1). Alternatively, the
aminal functional group of the diaza-adamantane derivatives
2 could be directly functionalized, to yield compounds 5-7
(Scheme 1). This synthetic route is two steps shorter, but the
functionalization step provided the final products in lower
yields (17-33% yield). It was applied to the fluorinated aryl
derivatives 5e, 6e,f and 7e,f.
o
maintained at 37 C in a humidified 5% CO2 atmosphere for
24 h. Cells were washed with PBS and centrifuged at 300 x
g. The pellet was resuspended in 500 ꢀl PBS, and 2 ꢀl of 1
o
mg/mL JC-1 reagent was added for 20 min at 37 C in the
dark. Cells were washed with PBS and analyzed for apopto-
sis using a flow cytometer.
Some of the ketone starting materials 1 were not com-
mercially available, and therefore had to be synthesized
(Scheme 2). They were prepared either by reduction of an
unsaturated analog (Scheme 2a) or by a one-pot oxidation-
condensation-reduction reaction sequence between acetone
and an alcohol, catalyzed by an iridium complex (Scheme
2b) [38].
Caspase-3,7 Activity
Enzyme activity was measured using the carboxyfluo-
rescein FLICA apoptosis detection kit caspase assay from
Immunochemistry technologies, LLC with FAM-DEVD-
FMK as a substrate, according to the manufacturer’s proto-
cols. B16-F10 cells (6.5 x 105) were plated in 100 mm cul-
ture dishes and grown for 24 h. 7e (30 ꢀM) and 8e (75 ꢀM)
were added in RPMI-1640 complete media for 24 h. The
medium was collected and the cells were washed with cold
PBS. The cells were scraped and then washed twice by cen-
trifugation at 500 x g for 5 min. The cell pellet was resus-
pended in 300 ꢀl RPMI-1640 medium supplemented with 2
ꢀM of 30 x FLICA solution and incubated for 1 h at 37 oC in
the dark. Cells were washed with wash buffer and analyzed
for apoptosis using a flow cytometer.
Pharmacology
Cytotoxic Activity
Representatives of the four families of functionalized
1,3-diaza-adamantan-6-ones 5-8 were tested in vitro for pro-
liferation inhibition of mouse B16-F10 melanoma cells. The
test is based on the MTT assay for mitochondrial dehydro-
genases activity of live cells. The 1,3-diaza-2-thia-
adamantan-6-one derivatives 5 and 6 showed poor anti-
melanoma activity (IC50 > 200 ꢀM). On the other hand, both
acid chloride 7 and acid 8 derivatives of 1,3-diaza-2-
phospha-adamantan-6-one exhibited significant anti-
melanoma activity, with IC50 values in the 10-60 ꢀM range
(Fig. 1).
Flow Cytometry
B16-F10 cells (7 x 105) were seeded in 100 mm culture
dishes, and allowed to attach overnight. The medium was
replaced with fresh complete medium containing the desired
concentration of 7e (20 - 60 ꢀM). Cells were incubated for
o
Effect of 1,3-diaza-adamantan-6-ones on Cell Cycle
24 h at 37 C, washed with PBS and centrifuged at 300 x g.
Both the cells growing as a monolayer (harvested by scrap-
ing) and those floating in the medium were collected. The
pellet was stained with 2ꢀl PI solution for 15 min, and ana-
lyzed using a flow cytometer.
To gain further insight into the mechanism by which cell
reduction is achieved, we applied fluorescence-activated cell
sorting (FACS) analysis to study the effect of one of the
compounds on cell cycle distribution (Fig. 2). A 24-h incu-
bation of B16-F10 cells with 20 ꢀM 7e induced enrichment
of cells in the G2/M phase. Higher doses of 7e (up to 60 ꢀM)
induced a profound sub-G1 peak at the expense of cells at all
other phases.
PI solution – To a mixture of 3 mL detergent 1% NP-40
and 15 mg RNase was added 10 mL water. The solution was
o
stirred at 100 C for 15 min. To the solution was added 2.5
mg PI (propidium iodide) and 37 mL water. The solution
was stored at 4 oC in the dark.
Induction of Apoptosis
RESULTS
Chemistry
We selected compounds 7e and 8e (5,7-dibenzyl-2-
chloro-1,3-diaza-2-phospha-adamantan-6-one 2-oxide and
5,7-dibenzyl-2-hydroxy-1,3-diaza-2-phospha-adamantan-6-
one 2-oxide, respectively), representing the two families of
the acid chloride and acid derivatives of 1,3-diaza-2-
phospha-adamantane, to further investigate the effect of the
1,3-diaza-adamantane compounds on the melanoma cells.
These were among the best proliferation inhibitors (IC50 val-
ues of 25 ꢀM and 60 ꢀM, respectively), and they bear the
same 5,7-dibenzyl substitution pattern so the effect of the 2-
functional group could be directly evaluated.
1,3-diaza-adamantanones can be obtained via a quadruple
Mannich reaction between a ketone, an aldehyde and ammo-
nia [31]. A practical and efficient procedure involves the
interaction of a ketone with hexamethylenetetramine [32].
Thus, a small library of six 5,7-dialkyl-1,3-diaza-adamantan-
6-ones 2a-f was prepared in this way in moderate to excel-
lent (45-95%) yields (Scheme 1). These compounds were
transformed into the corresponding 1,5-dialkyl-3,7-diAlloc-

32 Medicinal Chemistry, 2014, Vol. 10, No. 1
Sharabi-Ronen et al.
Alloc
NH
NH
N
N
N
O
Alloc
iii
i
ii
N
N
N
R
N
R
R
R
R
N
R
O
R
O
R
O
1
3
4
2
O
N
S
vii
iv
N
R
30%
88-100%
R
R
R
O
5
O
N
O
S
viii
v
N
R
17%
59-85%
O
6
R = n-butyl
n-pentyl
phenyl
(a)
(b)
(c)
(d)
(e)
(f)
O
N
Cl
P
ix
vi
N
R
benzyl
31-33%
82-100%
4-CF3-Ph-CH2
O
7
3,5-(CF3)2-Ph-CH2
86-100%
x
O
N
O-
P
N
R
R
O
8
Scheme (1). Synthetic scheme for the library of 1,3-diaza-2-functionalized-adamantan-6-one derivatives 5-8. (i) AcOH (cat), EtOH, reflux,
15 h. (ii) allyl chloroformate, CHCl3, rt, 5 days. (iii) AcOH, Pd(PPh3)4, Bu3SnH, CHCl3, rt, 4 h. (iv) SOCl2, Et3N, CHCl3, rt, 5 h. (v) SO2Cl2,
Et3N, CHCl3, rt, 4 h. (vi) POCl3, Et3N, CHCl3, rt, 5 h. (vii) SOCl2, pyridine, CH3CN, rt, 15 h. (viii) SO2Cl2, pyridine, CH3CN, rt, 15 h. (ix)
POCl3, pyridine, CH3CN, rt, 15 h. (x) NaOH (2 M, aq), THF, reflux, 15 h.
a
O
O
5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazolocar-
bocyanin iodide (JC-1 reagent) [39], and fluorescent-labeled
inhibitor of caspases (FLICA)-based quantification of in-
duced caspase 3 and 7 activity. Fig. (3) shows the effect of
24 h treatment of B16-F10 melanoma cells with compounds
7e (at 25 and 30 ꢀM) and 8e (at 50-75 ꢀM). Under the spe-
cific conditions employed, 7e at 30 ꢀM induced 28% apop-
tosis, whereas most of the cells (82%) underwent either
early- or late apoptosis when incubated with 75 ꢀM of 8e.
Identical results were obtained from the JC-1 experiment,
quantifying the percentage of cells with low membrane po-
tential (apoptotic cells) (Fig. 4).
i
Ph
Ar
Ph
Ar
Ph
Ph
1d
b
O
O
ii
ArCH2OH
1
Ar = 4-CF3-Ph
(e)
3,5-(CF3)2-Ph (f)
Scheme (2). Synthesis of ketone 1d-f starting materials. (i) H2 5
atm, Pd-C 10%, AcOH, EtOAc, 7 h. (ii) [IrCl(cod)]2, PPh3, KOH,
100 ºC, 5-7 h.
Apoptotic cell death may be initiated through the death
receptor (extrinsic) pathway, involving caspase 8 activation
[40], or through the mitochondrial (intrinsic) pathway and
caspase 9 activation [41]. Both pathways lead to activation
of caspases 3, 6, and 7 [42]. Thus, the effect of 1,3-diaza-2-
phospha-adamantan-6-ones 7e and 8e on B16-F10 melanoma
We carried out three sets of experiments to assess apop-
totic cell death: quantification of apoptotic cells by the an-
nexin V-FITC/PI assay, identification of mitochondrial
membrane potential changes by the fluorescent cationic dye

Diaza-adamantanones as Anti-melanoma Compounds
Medicinal Chemistry, 2014, Vol. 10, No. 1 33
100
90
80
70
60
50
40
30
20
10
0
5d 190
5c 340
6c 240
6b >800
0
100 200 300 400 500 600 700 800 900
Concentration (μM)
100
90
80
70
60
50
40
30
20
10
0
7d 20
7f 26
7e 25
7a 12
0
10
20
30
40
100
90
80
70
60
50
40
30
20
10
0
8e 60
8c 950
0
500
1000
1500
Concentration (μM)
Fig. (1). Inhibition of melanoma B16-F10 cells proliferation by 1,3-aza-2-functionalized-adamantan-6-ones 5-8. B16-F10 cells (5 x 103)
were grown in a 96-well microtiter plate for 24 h, and then incubated with the 1,3-diaza-adamantane compounds (10-1000 mM in DMSO) at
final concentration of 10-1000 ꢀM. After 24 h, cell proliferation was measured by a modified MTT assay. Data is presented as % prolifera-
tion inhibition in comparison with control cells treated with vehicle only (0.1% DMSO). IC50 values are listed in the graphs. The experimen-
tal points are presented as an average S.D. of three independent experiments. The lines are for clarity only.
were transferred either directly or via a short sequence of
aminal opening and amine protection / deprotection / func-
tionalization to the target compounds. Thus, a library of 1,3-
diaza-adamantan-6-one compounds, substituted at the 5 and
7 positions with alkyl or aryl groups and functionalized at
the 2 position was prepared. The 2-functional groups include
sulfurous amide (N2SO, 5), sulfamide (N2SO2, 6), phos-
phorodiamidic chloride (N2POCl, 7), and phosphorodiamidic
acid (N2PO2H, 8) (Fig. 1). The simplicity and the high yield
of the reactions make the presented synthetic methodology
suitable for the preparation of combinatorial libraries of 1,3-
diaza-adamantan-6-ones, with varying functional groups and
substituents.
cells was also evaluated by measuring the induction of
caspases 3 and 7 activity. Thus, 24 h incubation of the mela-
noma cells with 30 ꢀM of 7e lead to increased caspases ac-
tivity in 56% of the cells. Likewise, 24 h incubation with 8e
(75 ꢀM) induced caspase activity in 67% of the cells (Fig.
5).
DISCUSSION
Chemistry
A one-pot quadruple Mannich reaction afforded 5,7-
dialkyl/aryl-1,3-diaza-adamantan-6-one compounds that

34 Medicinal Chemistry, 2014, Vol. 10, No. 1
Sharabi-Ronen et al.
20μM
Control
7e
Sub-G1
G2/M
7e
7e
30μM
60μM
DNA content
Fig. (2). The effect of 1,3-diaza-2-phospha-adamantan-6-one 7e on cell-cycle distribution. B16-F10 cells were incubated with 7e (20, 30 and
60 ꢀM) for 24 h, and analyzed by DNA flow cytometry. The histograms show number of cells per channel (vertical axis) vs. DNA content
(horizontal axis). The values indicate the percentage of cells in the indicated phases of the cell cycle. The data shown are representative of
three independent experiments with similar results.
Pharmacology
potential assessment and quantification of caspases activa-
tion. Apoptosis was also demonstrated by changes in the cell
morphology (data not shown).
Malignant melanoma is a highly aggressive tumor that
frequently resists chemotherapy. Poor response, population
selectivity, limited response duration and other limitations of
the current drugs emphasize the urgent need to develop new
effective anti-melanoma drugs. Thus, in light of the activity
of similar compounds in other types of cancer [29], we tested
the in vitro anti-melanoma activity of the library of com-
pounds we synthesized on mouse B16-F10 melanoma cell
line. The library can be divided into two groups: the group of
1,3-diaza-2-thia-adamantan-6-one derivatives 5 and 6 exhib-
ited very poor anti-melanoma activity (IC50 > 200 ꢀM). On
the other hand, the two families of 2-phospha analogs 7
(phosphoryl chloride) and 8 (phosphate) exhibited much
better proliferation inhibition activity, with IC50 values as
low as 10 ꢀM. Two compounds, 7e and 8e, were selected for
mechanistic studies, since they vary in their functional group
at the 2 position, but share the same 5,7 substitution. The
inhibitory activity of 7e is due to G2/M arrest of the cell cy-
cle. The cell cycle arrest induces apoptosis, as demonstrated
by a set of various experiments (for both 7e and 8e), includ-
ing specific apoptotic staining, mitochondrial membrane
It is too early to speculate about the molecular mecha-
nisms underlying the cytotoxic activity of the 1,3-diaza-
adamantan-6-one derivatives. On one hand, various adaman-
tane derivatives act as sodium [26] and potassium [43] chan-
nel blockers and as receptor ligands [27]. On the other hand,
the diaza-adamantanone derivatives investigated in this
study, especially the phosphorodiamidic chloride derivatives
7, are hydrophobic molecules bearing an electrophilic func-
tionality. As such they are reminiscent of some sesquiterpene
lactones that exhibit anti-melanoma activity by modulating
the concentration of cell cycle regulatory proteins and reduc-
ing the expression of cell survival proteins [44]. Further
studies will shed light on the operative mechanisms. Future
studies of the interaction of the new compounds with their
biological target at a molecular level may also explain the
large difference in the activity of the four functional groups
presented in this study. At this stage it is not clear whether
this difference arises from different uptake by the cells or by

Diaza-adamantanones as Anti-melanoma Compounds
Medicinal Chemistry, 2014, Vol. 10, No. 1 35
a
Control
7e (25 μM)
7e (30 μM)
0.05%
7%
16%
Late apoptosis
Early apoptosis
0.01%
4%
12%
FL1-H
FL1-H
FL1-H
ꢀ
ꢀ
ꢀ
ꢁ
b
8e (50 μM)
8e (60 μM)
8e (75 μM)
18%
49%
76%
13%
16%
13%
FL1-H
FL1-H
FL1-H
Fig. (3). Annexin V-FITC binding and propidium iodide uptake induced by 1,3-diaza-2-phospha-adamantan-6-ones 7e and 8e. B16-F10 cells
were treated with (a) compound 7e (25 and 30 ꢀM) and (b) compound 8e (50, 60 and 75 ꢀM) for 24 h, stained with Annexin V-FITC and
propidium iodide, and analyzed by flow cytometry. The control cells were incubated with the vehicle (0.1% DMSO). The horizontal (FL1-H)
and vertical (FL2-H) axes represent labeling with Annexin V-FITC and PI, respectively. The data shown are representative of three independ-
ent experiments with similar results.
a
Control
20
7e (20 ꢀꢁꢂ
7e ꢃꢄꢅꢆꢀꢁꢂ
30
7e ꢃ ꢅꢆꢀꢁꢂ
ꢀ
M
25
ꢀ
M
ꢀ
M
40
ꢀM
95%
90%
73%
1%
5%
10%
27%
99%
ꢀ
Treatment 9f
b
8e (50 ꢀM)
8e (60 ꢀꢁꢂꢆ
8e ꢃꢇꢈ ꢀꢁꢂ
8e ꢃꢉꢅꢅꢆꢀꢁꢂ
50
ꢀ
M
60
ꢀ
M
75
ꢀ
M
100
ꢀM
83%
42%
11%
1%
17%
58%
89%
99%
Fig. (4). The effect of 1,3-diaza-2-phospha-adamantan-6-ones 7e and 8e on mitochondrial membrane potential. B16-F10 cells were treated
with (a) compound 7e (20-40 ꢀM) and (b) compound 8e (50-100 ꢀM) for 24 h to follow the extent of apoptosis by determination of mito-
chondrial membrane potential using JC-1 reagent. The control cells were incubated with the vehicle (0.1% DMSO). Cells were analyzed on a
FACScan cytometer. Dot plots of red (FL2-H) vs. green fluorescence (FL1-H) show live cells with intact mitochondrial membrane potential
and dead cells with lost mitochondrial potential, respectively. The data shown are representative of three independent experiments with simi-
lar results. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper).

36 Medicinal Chemistry, 2014, Vol. 10, No. 1
Sharabi-Ronen et al.
(ꢂ
8e
7e
Fig. (5). The effect of 1,3-diaza-2-phospha-adamantan-6-ones 7e and 8e on caspases 3 and 7 activities. B16-F10 Cells were incubated with 30
ꢀM 7e (right) and 75 ꢀM 8e (left) for 24 h and their caspase 3 and 7 activities were measured. The control cells were incubated with the vehi-
cle (0.1% DMSO). Enzymatic activity was determined with FAM-DEVD-FMK FLICA reagent and analyzed by flow cytometry. The fre-
quency of events (Y axis) vs. fluorescence intensity (X axis) graph shows 2 peaks: caspase negative cells occur to the left (unlabeled cells)
whereas caspase positive cells lay within the region in the right. The data shown are representative of three independent experiments with
similar results.
by DNA methylation: insights from transcriptomic studies. Car-
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CONFLICT OF INTEREST
The authors confirm that this article content has no
conflict of interest.
ACKNOWLEDGEMENTS
Partial support from the Raoul Wallenberg Chair for
Immunological Chemistry is gratefully acknowledged.
ABBREVIATIONS
FACS = Fluorescence-activated cell sorting
FITC = Fluorescein isothiocyanate
FLICA = Fluorescent-labeled inhibitor of caspases
[10]
[11]
JC-1
MTT
PI
= 5,5',6,6'-tetrachloro-1,1',3,3'- tetraethyl-
benzimidazolylcarbocyanine iodide
[12]
[13]
= 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-
zolium bromide
= Propidium iodide.
[14]
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Received: December 23, 2012
Revised: April 22, 2013
Accepted: April 22, 2013