Synthesis and potent antitumor activities of novel 1,3,5-cis,cis- triaminocyclohexane N-pyridyl derivatives

Article abstract of DOI:10.1021/jm040076w

The iron chelator N,N′,N″-tris(2-pyridylmethyl)-cis,cis-1,3,5- triaminocyclohexane (tachpyr) was recently reported to display potent antitumor activity. The present study was focused on identifying an adequate bifunctional version of tachpyr as a lead compound for use in antibody-targeted iron depletion tumor therapy. Preparation of tachpyr derivatives having a side chain is reported, and their cytotoxic activity is evaluated in the HeLa cell line. The observed cytotoxity appears dependent on the functionalization site of the tachpyr employed for introducing the protein conjugation. Tachpyr derivatives 14 and 15 having a side chain introduced into the 5-position of the pyridyl ring display more potent cytotoxicity than tachpyr derivatives 7 and 8 having a side chain introduced onto one of the secondary amines. BOC-protected tachpyr derivative 14 exhibited the most potent cytotoxicity against this cancer cell line, which was reasonably comparable to the parent tachpyr. Tachpyr derivative 14 was further converted into bifunctional tachpyr 17 possessing a maleimide linker for conjugation with thiolated monoclonal antibodies (mAbs).

Full text of DOI:10.1021/jm040076w

5230
J . Med. Chem. 2004, 47, 5230-5234
Syn th esis a n d P oten t An titu m or Activities of Novel
1,3,5-cis,cis-Tr ia m in ocycloh exa n e N-P yr id yl Der iva tives
Hyun-Soon Chong,*,† Suzy V. Torti,‡,§ Rong Ma,‡,§ Frank M. Torti,§, and Martin W. Brechbiel†
Chemistry Section, National Cancer Institute, NIH, Bethesda, Maryland, and Departments of Cancer Biology and Biochemistry
and the Comprehensive Cancer Center, Wake Forest University, Winston Salem, North Carolina
Received March 30, 2004
The iron chelator N,N′,N′′-tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane (tachpyr) was
recently reported to display potent antitumor activity. The present study was focused on
identifying an adequate bifunctional version of tachpyr as a lead compound for use in antibody-
targeted iron depletion tumor therapy. Preparation of tachpyr derivatives having a side chain
is reported, and their cytotoxic activity is evaluated in the HeLa cell line. The observed cytotoxity
appears dependent on the functionalization site of the tachpyr employed for introducing the
protein conjugation. Tachpyr derivatives 14 and 15 having a side chain introduced into the
5-position of the pyridyl ring display more potent cytotoxicity than tachpyr derivatives 7 and
8 having a side chain introduced onto one of the secondary amines. BOC-protected tachpyr
derivative 14 exhibited the most potent cytotoxicity against this cancer cell line, which was
reasonably comparable to the parent tachpyr. Tachpyr derivative 14 was further converted
into bifunctional tachpyr 17 possessing a maleimide linker for conjugation with thiolated
monoclonal antibodies (mAbs).
In tr od u ction
as compared to the tumors with the p53 gene.8 Consid-
ering that ∼50% of human tumors are deficient in the
p53 gene,8d tachpyr possesses great promise not only
as a single antitumor agent but for use in combination
with other chemotherapeutic agents.
The great promise of iron depletion as a cancer
treatment strategy1 has stimulated research on devel-
opment of iron chelators as anticancer agents.2 Among
the iron chelators being evaluated or employed as
antitumor agents are desferrioxmine (DFO),3 Triapine,4
and pyridoxal isonicotinoyl hydrazone (PIH) deriva-
tives.5 These iron chelators are known to cause cellular
iron depletion and exhibit potent cytotoxic activities on
numerous cancer cells including leukemia, neuroblas-
toma, hepatoma xenografts, and lymphoma.3-5 Many
studies to investigate mechanisms for the marked
inhibitory activities of the iron chelators on cancer cells
have been carried out. One mechanism may involve the
inhibition of key iron-dependent enzymes. For example,
DFO3d and Triapine4b have been shown to inhibit
ribonucleotide reductase, an iron-dependent enzyme
overexpressed in some tumor tissues.1b,4a
Our research has been focused on developing potent
iron chelators for use in iron depletion tumor therapy.
Recently, we reported that a novel iron chelator, N,N′,N′′-
tris(2-pyridylmethyl)-cis,cis-1,3,5-triaminocyclohexane
(tachpyr, 3),6 exhibits antitumor effects on cultured
cancer cells.7 Iron complexes of tachpyr were nontoxic,
suggesting that the capacity to bind metals is important
to the cytotoxic activity of this chelator.7a The most
striking finding was that tachpyr exhibited antitumor
activity irrespective of activation of the p53 tumor
suppressor gene.7c Tumors without the gene have been
reported to display significantly diminished sensitivity
to chemotherapeutic agents or angiogenesis inhibitors
A potential limitation of tachpyr as a clinically viable
anticancer drug that requires consideration is a rela-
tively short biological half-life. One approach to address
this limitation and enhance selective targeting is to link
tachpyr to a biomolecule such as a monoclonal antibody
(mAb). To test this approach, we proposed to design and
synthesize a bifunctional version of tachpyr possessing
a linker for conjugation with mAbs. We were particu-
larly interested in preparation of a bifunctional tachpyr
possessing an electrophilic maleimide group, which
would readily undergo Michael addition with a nucleo-
philic thiol group. Thus, a bifunctional tachpyr might
be readily conjugated to a selection of thiolated mAbs
using established conjugation chemistry to afford a
variety of antitumor conjugates that will possess a more
favorable biological half-life and allow for the selective
targeted delivery of tachpyr cytotoxicity.
As an ongoing effort to develop an adequate bifunc-
tional version of tachpyr without sacrificing its cytotox-
icity, we have designed a novel series of tachpyr ana-
logues substituted with different side chains and inves-
tigated their ability to act as cytotoxic agents. On the
basis of the observed cytotoxicity of the tachpyr ana-
logues described herein, we have proceeded to the design
and preparation of the bifunctional tachpyr 17 possess-
ing a maleimide moiety. Herein, we now report the syn-
thesis of the tachpyr analogues and the bifunctional
tachpyr 17. The cytotoxic activity of the tachpyr ana-
logues is measured in Hela cells using a (3-(4,5-dimeth-
ylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
assay and is compared to that of the parent compound,
tachpyr.
* To whom correspondence should be addressed. E-mail: Chong@
iit.edu. Phone: 312-567-3235. Fax: 312-567-3494. Mailing address:
3101 S. Dearborn St, Life Science 182, Chemistry Division, Biological,
Chemical and Physical Science Dept, Chicago, IL 60616.
† National Cancer Institute.
‡ Department of Cancer Biology, Wake Forest University.
§ The Comprehensive Cancer Center, Wake Forest University.
Department of Biochemistry, Wake Forest University.
10.1021/jm040076w CCC: $27.50 © 2004 American Chemical Society
Published on Web 09/03/2004

Synthesis and Potent Antitumor Activities
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5231
Sch em e 1
Sch em e 2
Resu lt a n d Discu ssion s
previously (Figure 1).11 The data in Figure 1 show that
Tachpyr analogues 7, 8, 14, and 15 possessing differ-
ent side chains designed for the present study are shown
in Scheme 1. Two different types of structural modifica-
tions to the tachpyr framework are sought to investigate
the effect of a side chain on the cytotoxic activity of
tachpyr. While analogues 7 and 8 possess a side chain
introduced onto one of the secondary amines of tachpyr,
analogues 14 and 15 possess a side chain introduced
into the 5-position of a pyridyl group in tachpyr.
Previously, we reported a convenient synthesis of tach
N-pyridyl derivatives via selective partial protection of
the amines in cis,cis-1,3,5-triaminocyclohexane (tach)
employing the bulky trityl (Tr) group.9 The synthetic
techniques reported therein have been directly applied
to the preparation of tachpyr analogues for the present
structure-activity relationship study. Tachpyr ana-
logues 7 and 8 were synthesized as previously published
(Scheme 2).9
The synthetic approach to tachpyr analogues 14 and
15 is shown in Scheme 3. Thus, commercially available
diester 9 was selectively reduced to 10 via a modification
of a previously published procedure.10 The hydroxyester
10 was reacted with 1,3-diaminopropane to afford the
intermediate 2-aminoethylamide 11, which was subse-
quently reacted with BOC-ON to afford carbamate 12.
BOC-protected 12 was oxidized (MnO2) to provide
aldehyde 13 in 92% yield. Reaction of 13 with bis-
(pyridylmethyl)tach 19 and subsequent reduction with
NaBH4 provided 14. The BOC group in 14 was removed
by treating with HCl to provide amine 15.
F igu r e 1. Effects of chelators 7 (1), 8 (9), 14 (]), 15 (2), and
tachpyr (b) on cell viability. Cells were incubated for 72 h with
chelators at various concentrations, and viability was assessed
as described in Materials and Methods. Each point represents
the mean and standard error of octuplicate cultures.
introduction of a side chain into the 5-position of the
pyridyl ring in tachpyr (14) is well tolerated and
resulted in minimal decrease in cytotoxicity as compared
with tachpyr. However, analogues 7 and 8, N-function-
alized at a secondary amine, display reduced cytotoxicity
as compared to C-functionalized tachpyr 14 and 15 over
the entire concentration range evaluated (Figure 1).
Nonetheless, all of the tachpyr analogues reported
herein are more cytotoxic agents than the previously
described tachpyr analogues 1 and 2 (Figure 2).9 We had
The cytotoxicity of tachpyr analogues 7, 8, 14, and
15 was evaluated using the HeLa cell line as described

5232 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Chong et al.
Sch em e 3
displayed the highest activity against HeLa cells and
was reasonably comparable to 3. Reactions were carried
out to convert 14 into bifunctional tachpyr 17 possessing
a maleimide linker for conjugation to thiolated antibody
(Scheme 3). Thus, reaction of 15 with the active ester
reagent 1612 followed by silica gel flash column chro-
matorgraphic purification provided 17 in 66% yield.
Although the antitumor efficacy of antibody conju-
gates based on the model compound 17 remains to be
determined, previous studies have demonstrated that
antibody conjugation can improve drug performance in
several ways, including a reduction in systemic toxicity,
increased potency, and prolonged half-life.13 Conjugated
mAbs have also shown a dramatic increase in antitumor
activity when used in combination therapy.14 We are
currently investigating biological activity of antitumor
conjugates of C-functionalized tachpyr 17, along with
N-functionalized tachpyr 189 prepared by reaction of 8
and 16 as previously described.
F igu r e 2. Effects of chelators 1 (1), 2 (0), 4 (O), 7 (∇), 14 (9),
and tachpyr (b) on cell viability. Cells were incubated for 72
h with chelators at various concentrations, and viability was
assessed as described in Materials and Methods. Each point
represents the mean and standard error of octuplicate cultures.
Con clu sion
We have prepared novel tachpyr analogues and
measured their cytotoxic activities on Hela cells. The
present study shows that choice of functionalization site
in tachpyr has meaningful impact on the cytotoxic
activity of tachpyr. The result of the cytotoxic assay
demonstrates that while a pyridyl ring moiety could be
substituted with a side chain without significant de-
crease in the cytotoxic activity of tachpyr, substitution
of one of three secondary amines with an analogous side
chain could result in decreased activity. Tachpyr deriva-
tives described herein will serve as the basis for our
continuing structure-activity relationship study of
these tach-derived iron chelators as antitumor drugs.
Future plans include an examination of impact on the
position of the pyridyl ring for functionalization on the
cytotoxicity. Studies are also ongoing with tach deriva-
tives having substituents other than pyridyl groups.
In sum, we have successfully prepared a bifunctional
tachpyr ligand 17 that possesses great promise as a
cancer therapeutic. Bifunctional ligand 17 might be
linked to a number of monoclonal antibodies targeting
previously reported that tachpyr analogues 1 and 2 ex-
hibited significantly decreased cytotoxicity as compared
to tachpyr 3.9 We had concluded from our prior study
that replacement of a pyridyl group in tachpyr 3 by ei-
ther hydrogen (1) or a propyl group (2) resulted in a
significant decrease in cytotoxicity and that all three
pyridyl groups of tachpyr should be retained in struc-
tural modifications of 3.9 The present study further
demonstrates that the presence of the six coordinating
groups, that is, three secondary amines and three pyri-
dylamines is required for maintaining the cytotoxic ac-
tivity of tachpyr analogues. It is interesting to note that
removal of the protecting groups (BOC or Phth) from 7
and 14, respectively, caused a slight decrease in cyto-
toxic activity. At this point, it is not clear whether the
distant primary naked amine might participate in iron
binding and somehow disturb the geometry and stability
of the six-coordinating binding sphere of tachpyr, thereby
leading to reduced cytotoxicity. Among all of tachpyr
derivatives, the BOC-protected tachpyr derivative 14

Synthesis and Potent Antitumor Activities
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21 5233
6.8 Hz, 2 H), 8.04 (d, J ) 8.1 Hz, 1 H), 8.37 (dd, J ) 2.1 Hz, 1
H), 9.27 (s, 1 H), 10.1 (s, 1 H); 13C NMR (CDCl3) δ 28.5 (q),
29.9 (t), 36.8 (t), 37.3 (t), 79.7 (s), 121.5 (d), 133.6 (s), 136.3
(d), 149.2 (d), 154.0 (s), 157.4 (s), 164.9 (s), 192.8 (s). HRMS
(positive ion FAB) Calcd for C15H21N3O4: [M + H]+ m/z
308.1622. Found: [M + H]+ m/z 308.1610. Anal. (C15H21N3O4‚
0.5H2O) C, H.
to various types of tumor cells to generate the first series
of antitumor conjugates for use in targeted iron deple-
tion tumor therapy. Studies pertaining to the develop-
ment of conjugation chemistry, tumor cell targeting, and
efficacy are ongoing and will be reported in due course.
Exp er im en ta l Section
(3-{[6-({3,5-Bis-[(p yr id in -2-ylm eth yl)-a m in o]-cycloh ex-
yla m in o}-m eth yl)-p yr id in e-3-ca r bon yl]-a m in o}-p r op yl)-
ca r ba m ic Acid ter t-Bu tyl Ester 14. To a solution of 1 (350
mg, 1.13 mmol) in benzene (25 mL) was added pyridinecar-
boxaldehyde (347 mg, 1.13 mmol). The resulting mixture was
refluxed using a Dean-Stark trap for 18 h. The reaction
mixture was cooled to the room temperature and evaporated
to dryness to provide pure imine as determined by NMR. The
obtained imine was dissolved in EtOH (10 mL) and reacted
with NaBH4 (43 mg, 1.13 mmol). The resulting mixture was
stirred at room temperature for 24 h and filtered, and the
filtrate was concentrated in vacuo. The residue was dissolved
in CH2Cl2 (15 mL), filtered again and dried (MgSO4), and the
filtrate was concentrated in vacuo to provide crude 14. The
crude product was suitable for next step or could be purified
via column chromatography on silica gel eluting with 0.8 mL
Et3N/15 mL MeOH/100 mL CH2Cl2. Pure 14 (771 mg, 93%)
was thereby obtained as a colorless oil: 1H NMR (CDCl3) δ
1.01 (dd, J ) 7.4 Hz, 4 H), 1.36 (s, 9 H), 1.54-1.65 (m, 2 H),
1.80 (s, 2 H), 2.17-2.21 (m, 3 H), 2.48-2.56 (m, 3 H), 3,14-
3.18 (m, 2 H), 3.35-3.45 (m, 2 H), 3.87 (s, 4 H), 3.92 (s, 2H),
5.26 (t, J ) 2.6 Hz, 1 H), 7.07 (dd, J ) 5.4 Hz, 2 H), 7.21 (d, J
) 7.6 Hz, 2 H), 7.31 (d, J ) 7.6 Hz, 1 H), 7.56 (dt, J ) 10 and
2.1 Hz, 2 H), 7.81 (s, 1 H), 8.06 (dd, J ) 2.4 Hz, 1 H), 8.46 (d,
J ) 5.2 Hz, 2 H), 8.96 (s, 1 H); 13C NMR (CDCl3) δ 27.8 (q),
29.3 (t), 35.7 (t), 36.4 (t), 39.6 (2C, t), 51.5 (t), 51.7 (t), 53.0
(2C, d), 121.1 (d), 121.3 (d), 121.7 (d), 127.9 (s), 134.8 (d), 135.8
(d), 147.5 (d), 148.5 (d), 156.5 (s), 159.0 (s), 162.1 (s), 165.1 (s).
HRMS (positive ion FAB) Calcd for C33H46N8O3: [M + Cs]+
m/z 735.2729. Found: [M + H]+ m/z 735.2747.
1
Gen er a l. H, 13C, and APT NMR spectra were obtained at
300 MHz in CDCl3 solution. Fast atom bombardment mass
spectra (FAB-MS) were obtained in the positive ion detection
mode. Elemental microanalysis was performed by Galbraith
Laboratories, Knoxville, TN.
6-H yd r oxym et h yl-n icot in ic Acid Met h yl E st er 10.
NaBH4 (149 mg, 3.95 mmol) was added to a slurry of pyridine
2,5-dicarboxylate (308 mg, 1.58 mmol) and CaCl2 (693 mg, 6.25
mmol) in THF (3.3 mL) and EtOH (6.7 mL) at 0 °C in portions.
The resulting mixture was stirred at 0 °C for 7 h, poured into
ice-water (5 mL), and extracted with CHCl3 (3 × 20 mL). The
combined organic layers were dried (MgSO4) and filtered, and
the filtrate was concentrated in vacuo to provide crude 10 (264
mg, 92%) as a colorless solid. The 1H NMR and 13C NMR
spectra of the material thereby obtained were essentially
identical to data reported previously for authentic 10.10 1H
NMR (CDCl3) δ 3.89 (s, 3 H), 4.42 (s, 1 H), 4.78 (s, 2 H), 7.38
(d, J ) 7.9 Hz, 1 H), 8.22 (dd, J ) 2.3 Hz, 1 H), 9.05 (s, 1 H);
13C NMR (CDCl3) δ 52.3 (q), 64.3 (t), 120.0 (d), 124.6 (s), 137.8
(d), 149.8 (d), 164.1 (s), 165.5 (s).
N-(3-Am in o-pr opyl)-6-h ydr oxym eth yl-n icotin am ide 11.
A mixture of 10 (6.68 g, 40 mmol), 1,3-diaminopropane (40 mL,
472 mmol), and NaCN (252 mg, 5.05 mmol) in MeOH (500
mL) was refluxed for 24 h. The resulting mixture was
concentrated in vacuo, and EtOH (100 mL) was added into
the residue. The resulting mixture was stirred for 5 min and
put into the freezer for 1 h. The mixture was filtered, and the
filtrate was concentrated in vacuo. The residue was dissolved
into CHCl3 (100 mL), and the mixture was stirred for 4 h at
room temperature and filtered. The filtrate was dried (MgSO4),
refiltered, and concentrated in vacuo to afford crude 11 (3.90
g, 43%): 1H NMR (CDCl3) δ 0.98-1.02 (m, 2 H), 1.90-1.98
(m, 2 H), 2.64-2.70 (m, 2 H), 3.95 (s, 2 H), 4.17 (s, 4 H), 6.84
(d, J ) 7.4 Hz, 1 H), 7.42 (dd, J ) 3.2 Hz, 1 H), 8.09 (s, 1 H);
13C NMR (CDCl3) δ 33.2 (t), 38.5 (t), 39.9 (t), 65.4 (t), 121.5
(d), 130.2 (s), 137.5 (d), 148.7 (d), 165.7 (s), 167.9 (s). The crude
product was used directly in the next step.
{3-[(6-H yd r oxym et h yl-p yr id in e-3-ca r b on yl)-a m in o]-
p r op yl}-ca r ba m ic Acid ter t-Bu tyl Ester 12. A solution of
BOC-ON (4.47 g, 18.2 mmol) in CH3CN (40 mL) was added
dropwise to a slurry of 11 (3.5 g, 16.5 mmol) in CH3CN (60
mL) over 1 h. The resulting mixture was stirred for 7 h at
room temperature. The solvent was evaporated, and the
residue was dissolved into ether (100 mL). The mixture was
washed with 20% NaOH (50 mL) and H2O (2 × 50 mL). The
ether layer was dried (MgSO4), filtered, and evaporated. The
residue was purified on silica gel chromatography eluted with
8% MeOH in CH2Cl2 to provide pure 12 (4.45 g, 87%) as a
colorless solid: 1H NMR (CDCl3) δ 1.44 (s, 9 H), 1.69-1.74
(m, 2 H), 3.26 (q, 2 H), 3.50 (q, 2 H), 4.80 (s, 2 H), 4.96 (t, J )
7.2 Hz, 1 H), 7.34 (d, J ) 6.9 Hz, 1 H), 7.77 (s, 1 H), 8.16 (d,
J ) 7.6 Hz, 1 H), 9.01 (s, 1 H); 13C NMR (CDCl3) δ 28.3 (q),
29.9 (t), 35.9 (t), 36.9 (t), 64.2 (t), 79.8 (s), 120.1 (d), 129.0 (s),
135.7 (d), 147.3 (d), 157.2 (s), 162.0 (s), 165.3 (s). HRMS
(positive ion FAB) Calcd for C15H23N3O1: [M + H]+ m/z
310.1778. Found: [M + H]+ m/z 310.1767. Anal. (C15H23N3O4‚
0.5H2O) C, H.
{3-[(6-F or m yl-p yr id in e-3-ca r b on yl)-a m in o]-p r op yl}-
ca r ba m ic Acid ter t-Bu tyl Ester 13. A mixture of 12 (1.78
g, 5.74 mmol) and MnO2 (4.69 g, 54 mmol) in CHCl3 (20 mL)
was refluxed for 5 h. The resulting mixture was filtered, and
the filtrate was concentrated in vacuo. The residue was
purified using silica gel column chromatography eluted with
5% MeOH in CH2Cl2 to provide pure 13 (1.69 g, 96%) as a
colorless solid: 1H NMR (CDCl3) δ 1.46 (s, 9 H), 1.70-1.78
(m, 2 H), 3.26-3.32 (m, 2 H), 3.52-3.58 (m, 2 H), 4.84 (t, J )
N-(3-Am in o-p r op yl)-6-({3,5-b is-[(p yr id in -2-ylm et h yl)-
a m in o]-cycloh exyla m in o}-m eth yl)-n icotin a m id e 15. Four
molar HCl in dioxane (5 mL) was slowly added to 14 (360 mg,
0.6 mmol) at 0 °C. The resulting mixture was warmed to room
temperature and stirred for 18 h. Ether (40 mL) was added
into the mixture, and the mixture was held in the freezer for
3 h. The tan solid was filtered and dissolved in H2O (10 mL).
The aqueous solution was adjusted to pH 7 with 5 M NaOH,
washed with CHCl3 (10 mL), and adjusted to pH 13. The
aqueous solution was evaporated, and the residue was treated
with CHCl3 (3 × 50 mL). The combined organic layers were
dried (MgSO4) and filtered, and the filtrate was concentrated
1
in vacuo to provide pure 15 (357 mg, 94%). H NMR (CDCl3)
δ 0.88 (dd, J ) 7.4 Hz, 4 H), 1.55-1.85 (m, 5 H), 2.08-2.18
(m, 3 H), 2.36-2.50 (m, 3 H), 2.72 (t, J ) 5.8 Hz, 2 H), 3,38-
3.45 (m, 2 H), 3.75 (s, 4 H), 3.81 (s, 2H), 7.07 (dd, J ) 6.4 Hz,
2 H), 7.13 (d, J ) 7.6 Hz, 2 H), 7.26 (d, J ) 7.6 Hz, 1 H), 7.46
(dt, J ) 8.7 and 2.1 Hz, 2 H), 7.93 (dd, J ) 2.5 Hz, 1 H), 8.36
(d, J ) 3.7 Hz, 2 H), 8.53 (s, 1 H), 8.80 (s, 1 H); 13C NMR
(CDCl3) δ 30.6 (t), 39.1 (t), 39.9 (2C, t), 40.4 (t), 51.8 (t), 52.0
(t), 53.3 (d), 121.4 (d), 121.5 (d), 121.9 (d), 128.3 (s), 135.1 (d),
136.1 (d), 147.4 (d), 148.7 (d), 159.3 (s), 162.3 (s), 165.1 (s).
HRMS (positive ion FAB) Calcd for C28H38N8O: [M + Cs]+ m/z
635.2223. Found: [M + Cs]+ m/z 635.2198.
6-({3,5-Bis-[(pyr idin -2-ylm eth yl)-am in o]-cycloh exylam i-
n o}-m et h yl)-N-{3-[4-(2,5-d ioxo-2,5-d ih yd r o-p yr r ol-1-yl)-
bu tyr yla m in o]-p r op yl}-n icotin a m id e 17. To a solution of
15 (120 mg, 0.24 mmol) in anhydrous DMF (4 mL) was added
active ester 16 (67 mg, 0.24 mmol). The resulting mixture was
stirred for 18 h at room temperature, and solvent was removed
via high-vacuum rotary evaporation at room temperature. The
residue was purified via neutral alumina flash chromatograpy
eluting with 8% methanol in CH2Cl2 to provide 17 (105 mg,
66%) as a colorless oil: 1H NMR (CDCl3) δ 1.18-1.34 (m, 4
H), 1.60-1.85 (m, 3 H), 1.90-1.99 (m, 3 H), 2.17-2.34 (m, 3
H), 2.53-2.82 (m, 5 H), 3.36 (q, 2 H), 3.50 (q, 2 H), 3.58 (t, 2
H), 3.98 (s, 4 H), 4.01 (s, 2 H), 6.52 (t, J ) 8.0 Hz, 1 H), 6.70

5234 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 21
Chong et al.
S. P. Triapine (3-aminopyridine-2-carboxaldehyde-thiosemicar-
bazone): a potent inhibitor of ribonucleotide reductase activity
with broad spectrum antitumor activity. Biochem. Pharmacol.
2000, 59, 983-991. (c) Cory, J . G.; Cory, A, H.; Rappa, G.; Lorico,
A.; Liu, M. C.; Lin, T. S.; Sartorelli, A. C. Inhibitors of ribo-
nucleotides reductase. Comparative effects of amino- and hy-
droxy substituted pyridne-2-carboxaldehyde thiosemicarbazones.
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(s, 2 H), 7.15 (ddd, J ) 11.2, 6, and 2.4 Hz, 2 H), 7.32 (d, 2 H),
7.38 (d, 1 H), 7.64 (dt, J ) 9.2 and 2 H), 7.87 (t, J ) 8.0 Hz, 1
H), 8.13 (dd, J ) 8 and 2.4 Hz, 1 H), 8.51 (d, J ) 6.0 Hz, 2 H),
9.04 (d, J ) 1.6 Hz, 1 H); 13C NMR (CDCl3) δ 24.7 (t), 29.3 (t),
33.5 (t), 35.9 (t), 36.0 (t), 37.13 (t), 37.93 (t), 51.6 (t), 51.77 (t),
51.79 (t), 53.40 (d), 53.46 (d), 122.1 (d), 122.4 (d), 122.7 (d),
128.8 (d), 134.2 (d), 135.7 (d), 136.7 (d), 148.2 (d), 149.2 (d),
157.8 (s), 161.4 (s), 165.6 (s), 170.9 (s), 172.9 (s); MS (positive
ion FAB) Calcd for C36H45N9O4: [M + H]+ m/z 668.36. Found:
[M + H]+ m/z 668.38.
Cytotoxicity Assa y. Hela cells were obtained from the
American Type Culture Collection and grown in a humidified
5% CO2 atmosphere at 37 °C in DME medium (Gibco BRL)
supplemented with 10% fetal bovine serum and penicillin/
streptomycin.
A total of 2 × 103 cells were plated in 96 well tissue culture
dishes and allowed to attach overnight before test compounds
were added. Six replicate cultures were used for each point.
After 72 h, viability was assessed using an MTT assay, in
which (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide is added to the medium and the formation of a
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Ack n ow led gm en t. We thank the structural mass
spectra group (Dr. Sonja Hess, NIDDK, Bethesda, MD)
for obtaining HRMS spectra of all the compounds. This
work was supported in part by grant No. DK 57781
(S.V.T.) from the National Institutes of Health.
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