Facile Synthesis of Pyridazine-based α-Helix Mimetrics

Article abstract of DOI:10.3987/COM-07-S(U)45

The synthesis of an amphiphilic, nonpeptidic scaffold that mimics the presentation of i, i+4, and i+7 residues of an α-helix is presented. The approach uses a pyridazine core, and minimizes the number of C-C bond forming reactions. The synthesis of this Urea-Pyridazine-Piperazine (UPP) scaffold is versatile and its synthesis makes it suitable for the preparationof small libraries of low-molecular-weight α-helix mimetics that can be targeted to specific protein/protein interactions.

Full text of DOI:10.3987/COM-07-S(U)45

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HETEROCYCLES, Vol. 73, 2007, pp. 661 - 671. © The Japan Institute of Heterocyclic Chemistry
Received, 30th June, 2007, Accepted, 20th August, 2007, Published online, 20th August, 2007. COM-07-S(U)45
FACILE SYNTHESIS OF PYRIDAZINE-BASED α-HELIX MIMETICS
Lionel Moisan, Trevor J. Dale, Naran Gombosuren, Shannon M. Biros,
Enrique Mann, Jun-Li Hou, Fernando P. Crisostomo, and Julius Rebek, Jr.*
The Skaggs Institute for Chemical Biology, The Scripps Research Institute, Mail
MB-26, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA, Fax:
(+1)858-784-2876, E-mail: jrebek@scripps.edu
Abstract – The synthesis of an amphiphilic, nonpeptidic scaffold that mimics the
presentation of i, i+4, and i+7 residues of an α-helix is presented. The approach
uses a pyridazine core, and minimizes the number of C-C bond forming reactions.
The synthesis of this Urea-Pyridazine-Piperazine (UPP) scaffold is versatile and
its synthesis makes it suitable for the preparation of small libraries of
low-molecular-weight α-helix mimetics that can be targeted to specific
protein/protein interactions.
INTRODUCTION
The α-helix is one of the most abundant secondary protein structures and is often a key feature for
protein-protein recognition. Side chains in positions i, i+3/i+4, i+7, and i+11 appear on the same face of
the helix are frequently crucial for the interaction.1 Hamilton and co-workers pioneered the synthesis of
non-peptidic α-helix mimetics based on terphenyl, terephthalamide, and oligopyridine scaffolds that
display side chains in a manner that closely resembles those in position i, i+4, and i+7 of an α-helix.2
They were shown to efficiently disrupt protein-protein interactions such as Bak/Bcl-XL,3 p53/HDM2,4
calmodulin/smooth muscle myosin light-chain kinase,5 and gp41 assembly.6 In our general efforts
towards the design of inhibitors of protein-protein interactions,7 we were interested in developing
methodology for structurally similar molecules featuring more hydrophilic components and a facile
synthetic route.8 In the present paper we describe the synthesis of a new class of low-molecular-weight
α-helix mimetics featuring a pyridazine ring and hydrophobic amino-acid side chains.
The target molecules are shown in Figure 1 along with an overlay with an α-helix (Figure 1a). The
scaffold mimics the position i, i+4, and i+7 of the α-helix. To ensure the water solubility of our inhibitors
we based our scaffold on a pyridazine ring. These molecules may be thought of as synthetic counterparts

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to amphiphilic α-helices; they are intended to present both a hydrophobic surface for recognition and a
hydrophilic “wet edge” that is rich in hydrogen bond donors and acceptors.
Figure 1. a) Overlay of 1 with a generic α-helix; b) General retrosynthetic approach of the UPP scaffold 1.
RESULTS AND DISCUSSION
The general retrosynthetic approach to these molecules is laid out in Figure 1b. The major disconnections
from the final urea-pyridazine-piperazine 1 (UPP) are made at the amide and urea bonds to give a
pyridazine diester 2, an amine 3, and a piperazine 4. This synthesis is modular, many amines are
commercially available as are a variety of Boc-protected 2-substituted piperazines presenting standard
hydrophobic side chains (i.e. benzyl, isobutyl, etc.). The central pyridazine ring is readily forged from the
inverse electron demand Diels-Alder reaction of dimethyl-1,2,4,5-tetrazine-dicarboxylate 5 and a suitable
dienophile.9
Tetrazine 5 was synthesized from ethyldiazoacetate following the procedure of Boger and co-workers10
and reacted with a range of dienophiles to introduce the side chain R2 and give the corresponding
pyridazines (Scheme 1). Three kinds of dienophiles were employed depending on the structure of the
desired R2 group. A few suitable alkynes such as 6 are commercially available and were used. Enamines
are also known to be good dienophiles for the cycloaddition with disubstituted tetrazines.9 Thus R2 = iPr
was introduced via the cycloaddition of the enamine 7 obtained by the condensation of isovaleraldehyde
and pyrrolidine following the Mannich procedure.11 This procedure is suitable to introduce short, low
molecular weight side-chains such as iPr for which the corresponding alkynes are often a gas and more
difficult to handle. In the case where R2 = CH2-BocIndole, methyl enol ether 8 was used as the dienophile.
The enol ether was obtained by methylenation of the corresponding methyl ester with Tebbe’s reagent12
and directly subjected to reaction with tetrazine 5 without any further purification. This procedure
allowed us to expand the scope of the possible side chains to the wide range of methyl esters

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663
commercially available, especially those derived from natural amino-acids.
CO2Me
CO2Me
R2
Dienophile Pyridazine Conditions Yield
MeO
N
dienophile
dioxane
90 oC
90 oC
rt
N
N
N
N
N
N
6
7
8
67%
67%
31%
2c
2a
2b
7
N
O
O
CO2Me
CO2Me
6
8
5
2
Scheme 1. Inverse electron demand Diels-Alder reaction of tetrazine 5 with various dienophiles.
Installation of the piperazine group was performed by using either a peptide coupling (Method A) or
direct coupling of the methyl ester function (Method B) (Scheme 2). A few examples were first carried
out with non substituted N-Boc protected piperazines (R3 = H). The corresponding final compounds
present only two side chains but provide clear NMR spectra for characterization. The synthesis was then
applied to substituted piperazines yielding compounds presenting three side chains for which the NMR
spectra are more complex due to the conformational isomers of the piperazine around the tertiary amide
bond. The 6-position methyl ester of the pyridazine 2a was selectively saponified using LiOH in a
THF/water mixture at 0 °C. The piperazine 4a (R3 = H) was coupled under standard peptide coupling
conditions with PyBroP (Method A) in good yields to give pyridazine-piperazine 9a. In the case of
pyridazines 9b,c the coupling with piperazines 4a or 4b (R3 = iBu) was performed directly using Weinreb
amidation13 in the presence of AlMe3 (Method B) to give only the desired regioisomer.
NH2
O
NH
O
N3
CO2Me
CO2Me
R2
3
6
Method A or B
R2
NH2-NH2
MeOH, rt
R2
R2
NaNO2, HCl
N
N
N
N
N
N
N
N
THF/H2O, 0 °C
R3
HN
CO2Me
R3
R3
R3
NBoc
O
O
O
N
N
N
4a
4b
R3 = H
R3 = iBu
NBoc
NBoc
NBoc
2a
2b
2c
R
R
2 = iPr
2 = BocInd
11
R2 = iBu
9a R2 = iPr, R3 = H (Method A, 58%)
9b R2 = BocInd, R3 = H (Method B, 61%)
10a
10b
10c
(88%)
(79%)
(82%)
9c
R
2 = iBu, R3 = iBu (Method B, 68%)
Method A or B
A : 1) LiOH, THF/H2O, 0 °C; 2) PyBroP, NEt3, CH2Cl2, rt.
B : AlMe3, CH2Cl2, reflux.
R1
R1
iPr
HN
HN
HN
HN
O
•
iBu
O
N
O
R
1-NH2
70 °C
R2
TFA
R2
R2
iAm
N
N
N
N
Ttoluene, rt
CH2Cl2, rt
toluene
N
N
MethioPr
BocInd
S
R3
R3
R3
NH
O
O
O
N
N
N
NBoc
NBoc
1a
1b
1c
1d
N
(97%)
(94%)
(73%)
(90%)
13a
13b
13c
R1 = iAm, R2 = iPr, R3 = H (52%)
12
R1 = MethioPr, R2 = iPr, R3 = H (52%)
1 = iAm, R2 = BocInd, R3 = H (31%)
O
R
O
13d R1 = iAm, R2 = iBu, R3 = iBu (25%)
Scheme 2. Synthesis of the UPP scaffold 1.

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To install the urea function in the 3-position, the remaining methyl ester was first converted to the acyl
hydrazide 10a-c under mild conditions using an excess of hydrazine in methanol. The acylhydrazide
function was then diazotized to the acyl azide 11 using the Curtius method (sodium nitrite in acidic
conditions).14 Acyl azides were not purified and directly transformed to the corresponding isocyanates 12
by heating at 70 oC. Ureas 13a-d were obtained by trapping these isocyanates with various amines. Final
removal of the N-Boc group was accomplished using TFA, to give the desired UPP 1a-d which present
three side chains to mimic the i, i+4, i+7 positions of an α-helix.
CONCLUSIONS
In conclusion, the synthesis of the desired α-helix mimetics has been performed in few steps. While
specific derivatives are prepared and disclosed here, the methodology reported is applicable for a broader,
more general decoration of the scaffold to provide a diversity of compounds. A library of over thirty
compounds that bear common hydrophobic amino-acid side chains was synthesized with this strategy and
it is currently being evaluated for the inhibition of various protein-protein interactions.
EXPERIMENTAL
Solvents and reagents were of reagent-grade, purchased from commercial suppliers, and used without
further purification unless otherwise stated. Substituted NBoc-protected piperazines were purchased from
Anaspec. Thin-layer chromatography (TLC) was performed on Kieselgel 60 F254 coated plates (Merck).
13
1H and C NMR spectra were recorded on Bruker 250 MHz, 300 MHz, or 600 MHz spectrometers.
Chemical shifts are expressed in ppm (δ), referenced to the protio impurity of the solvent as internal
standard for 1H and 13C nuclei. High resolution mass spectra were recorded on an Agilent ESI-TOF mass
spectrometer by Scripps Center for Mass Spectrometry.
Dimethyl 4-isopropylpyridazine-3,6-dicarboxylate (2a): To a stirred solution of isovaleraldehyde (1
mL, 9.2 mmol) and K2CO3 (1.5 g, 11.1 mmol) in CH2Cl2 (50 mL) was added dropwise over 1 h
pyrrolidine (780 μL, 9.3 mmol) at 0 °C. The mixture was vigorously stirred under nitrogen for 18 h. The
mixture was then filtered and the solvent removed in vacuo. The crude enamine was then used directly
without any further purification and added to a solution of tetrazine dimethylester 5 (1 g, 5.05 mmol) in
dioxane (50 mL) at 0 °C. The mixture was stirred for 4 h at rt, heated at 90 °C for 16 h, and then
concentrated in vacuo. The crude residue was purified on silica (CH2Cl2/AcOEt 1/0 to 9/1) to afford
compound 2a (800 mg, 3.36 mmol, 67%) as a pale yellow solid; 1H NMR: (250 MHz, CDCl3) δ 8.15 (s,
13
1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.38 (hept, J = 6.9 Hz, 1H), 1.27 (d, J = 6.8 Hz, 6H); C NMR: (62.5
MHz, CDCl3) δ 165.2, 164.2, 154.2, 151.8, 148.2, 125.5, 53.4, 53.2, 28.8, 22.5; HRMS: (ESI-TOF)

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C11H14N2O4H+ expected: 239.1026. found: 239.1024.
Dimethyl 4-((1-(tert-butoxycarbonyl)-1H-indol-3-yl)methyl)pyridazine-3,6-dicarboxylate (2b): To a
solution of methyl (1-tert-butyloxycarbonyl-indol-3-yl)acetate15 (3 g, 10 mmol) in THF (30 mL) cooled at
-40 ºC was added Tebbe’s reagent (22 mL, 1.1 eq, 0.5 M in toluene) after 30 min temperature was raised
to ambient over a period of 2 h. The mixture was then cooled to -10 ºC and the reaction was quenched by
the dropwise addition of NaOH (2.4 mL, 2 M solution). Reaction mixture was then allowed to warm to rt
The dark green solution was then diluted with excess ether and filtered through a plug of celite. Solvent
was removed under reduced pressure and the crude residue was directly diluted in dioxane (40 mL) and
added to a solution of tetrazine 5 (2 g, 10 mmol) in dioxane (10 mL). After 18 h at rt, the volatiles were
removed and the crude residue was purified on silica gel (CH2Cl2/AcOEt 1/0 to 15/1) to afford pyridazine
1
2b (1.3 g, 3 mmol, 31%) as a solid; H NMR: (600 MHz, CDCl3) δ 8.14 (bs, 1H), 8.02 (s, 1H), 7.46 (s,
13
1H), 7.35 (m, 1H), 7.30 (m, 1H), 7.21 (m, 1H), 4.40 (s, 2H), 4.07 (s, 3H), 4.03 (s, 3H), 1.69 (s, 9H); C
NMR: (150 MHz, CDCl3) δ 165.1, 164.0, 153.7, 151.8, 141.3, 129.3, 128.6, 124.9, 124.8, 122.8, 118.1,
116.7, 115.5, 115.1, 84.0, 53.4, 28.1, 27.1; HRMS: (ESI-TOF) C22H23N3O6H+ expected: 426.1660. found:
426.1670.
Dimethyl 4-isobutylpyridazine-3,6-dicarboxylate (2c): To a solution of tetrazine 5 (500 mg, 2.52
mmol) in anhydrous 1,4-dioxane (12.5 mL) was added 4-methylpentyne (234 mg, 2.85 mmol), the
reaction vessel was then sealed and heated to 90 °C for 18 h. The volatiles were removed under reduced
pressure, and the crude product was purified by silica gel chromatography (CH2Cl2/AcOEt 95/5) to give
2c (423 mg, 67% yield) as a yellow solid. 1H NMR: (600 MHz, CDCl3) δ 8.07 (s, 1H), 4.10 (s, 3H), 4.07
(s, 3H), 2.83 (d, J = 7.3 Hz, 2H), 1.97 (m, 1H), 0.95 (d, J = 6.6 Hz, 6H); 13C NMR: (150 MHz, CDCl3) δ
165.4, 164.5, 154.8, 151.6, 142.6, 129.9, 53.7, 53.5, 40.8, 29.7, 22.5; MS: (ESI-TOF) C12H16N2O4H+
expected: 253.1183, found 253.1187.
Procedures for the coupling of pyridazine with piperazines:
Method A:
Methyl 6-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-4-isopropylpyridazine-3-carboxylate (9a):
To a stirring solution of pyridazine 2a (536 mg, 2.25 mmol) in THF (10 mL) at 0 °C was added dropwise
a cold solution of LiOH (monohydrate, 99 mg, 2.36 mmol) in water (5 mL). The reaction was stirred at
0 °C until disappearance of the starting material on TLC. The pH was then made acidic (pH 1-2) with
careful addition of a 3% HCl solution. The solution was extracted with AcOEt (3x30 mL), the fractions
were combined, dried with Na2SO4, filtered and the solvent was removed in vacuo. The desired
monosaponified pyridazine was obtained as a pale yellow solid. The carboxylic acid was then directly
involved in the next step without any further purification. To a solution of the acid (312 mg, 1.39 mmol)

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in CH2Cl2 (7 mL) was added NEt3 (387 μL, 2.78 mmol), N-Boc-piperazine (285 mg, 1.53 mmol) and
PyBroP (714 mg, 1.53 mmol). After 18 h under nitrogen, the reaction mixture was diluted with CH2Cl2
(50 mL) and washed with a solution of HCl (0.1 M, 10 mL) and then a saturated aqueous solution of
NaHCO3 (10 mL). The organic layer was then dried with Na2SO4, filtered and concentrated under
reduced pressure. The crude residue was purified by column chromatography (Hexane/AcOEt 1/0 to 6/4)
and the desired piperazine adduct 9a (516 mg, 1.31 mmol, 58%) was obtained as a pale yellow foam; 1H
NMR: (250 MHz, CDCl3) Mixture of conformers δ 7.88 (s, 1H), 4.03 (s, 3H), 3.79 (m, 2H), 3.70 (m, 2H),
3.59-3.40 (m, 5H), 1.44 (s, 9H), 1.28 (d, J = 6.5 Hz, 6H); 13C NMR: (62.5 MHz, CDCl3) δ 165.1, 164.5,
156.8, 154.4, 152.8, 149.0, 125.8, 80.3, 53.2, 47.3, 43.6, 43.3 (broad), 42.7, 28.8, 28.2, 22.5; HRMS:
(ESI-TOF) C19H28N4O5H+ expected: 393.2132. found: 393.2126.
Method B:
To a solution of piperazine (1 equivalent) in anhydrous CH2Cl2 (5 mL) was added slowly AlMe3 (1
equivalent, 2.0 M solution in hexanes). The mixture was allowed to stir at rt for approximately 10 min. To
the aluminum-amide solution was added slowly a solution of pyridazine (1.05 equivalent) in anhydrous
CH2Cl2 (2.5 mL). The schlenk flask was sealed, and the yellow solution heated to 41 °C. After 24 h, the
now orange solution was cooled to room temperature, and quenched with slow addition of water (3 mL)
with vigorous stirring. The aqueous layer was extracted with CH2Cl2 (3x5 mL). The organic fractions
were collected, dried over MgSO4, and evaporated to dryness under reduced pressure. The crude residue
was purified by flash chromatography on silica gel.
tert-Butyl 3-((6-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-3-(methoxycarbonyl)pyridazin-4-
yl)methyl)-1H-indole-1-carboxylate (9b); Starting from 2b (398 mg, 0.93 mmol), column
Hexane/AcOEt 7/3, white solid, yield: 331 mg, (61%);1H-NMR (600 MHz, CDCl3) Mixture of
conformers δ 8.15 (bs, 1H), 7.67 (bs, 1H), 7.48 (bs, 1H), 7.33 (dd, J = 7.2, 8.4 Hz, 1H), 7.29 (d, J = 7.8
Hz, 1H), 7.19 (dd, J = 7.2, 8.4 Hz, 1H), 4.41 (s, 2H), 4.08 (s, 3H), 3.74 (bs, 2H), 3.63 (bs, 2H), 3.53 (bs,
2H), 3.48 (bs, 2H), 1.68 (s, 9H), 1.46 (s, 9H); 13C-NMR (150 MHz, CDCl3) δ: 165.0, 164.3, 156.9, 154.5,
152.2, 149.5, 142.2, 129.4, 128.8, 125.1, 124.9, 122.8, 118.6, 115.6, 115.0, 84.1, 80.4, 53.4, 47.4, 42.7,
28.4, 28.2, 27.2; HRMS: (ESI-TOF) C30H37N5O7H+ expected: 580.2766, found: 580.2759.
(S)-Methyl 6-(4-(tert-butoxycarbonyl)-3-isobutylpiperazine-1-carbonyl)-4-isobutylpyridazine-3-
carboxylate (9c); Starting from 2c (160 mg, 0.63 mmol), column Hexane/AcOEt 1/1, white foam, yield:
200 mg, (68%); 1H NMR: (600 MHz, CDCl3) Mixture of conformers δ 7.76 (s, 1H), 4.64 (m, 0.4H), 4.58
(m, 0.6H), 4.36 (m, 0.4H), 4.22 (m, 0.5H), 4.15 (m, 0.5H), 4.08 (m, 0.6H), 4.05 (s, 1.4H), 4.04 (s, 1.6H),
3.95 (m, 0.4H), 3.45 (m, 0.4H), 3.24 (m, 0.4H), 3.20-3.07 (m, 1.8H), 3.04 (m, 0.6H), 2.96 (m, 0.4H),
2.88-2.74 (m, 2H), 1.94 (m, 1H), 1.62-1.52 (m, 1H), 1.47 (s, 4.4H), 1.46 (s, 4.6H), 1.44 (m, 1H), 1.23 (m,

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13
1H), 0.96-0.91 (m, 9.5H), 0.82 (m, 2.5H); C NMR: (150 MHz, CDCl3) δ 165.0, 164.9, 156.5, 154.4,
153.1, 152.9, 143.1, 142.8, 129.9, 129.8, 80.2, 53.2, 50.2, 47.6, 45.8, 42.7, 40.6, 37.9, 29.3, 28.3, 24.6,
24.4, 22.8, 22.6, 22.4, 22.3, 22.2; HRMS: (ESI-TOF) C24H38N4O5H+ expected: 463.2915. found:
463.2915.
General procedure for the hydrazynolysis of the methyl ester pyridazines:
To a solution of pyridazine methyl ester (1 equivalent) in MeOH (30 mL) was added hydrazine
monohydrate (10 equivalents). The reaction was stirred for 20 h at rt under an atmosphere of nitrogen.
The volatiles were evaporated under reduced pressure, and the product was purified on silica gel.
tert-Butyl
4-(6-(hydrazinecarbonyl)-5-isopropylpyridazine-3-carbonyl)piperazine-1-carboxylate
(10a) : Starting from 9a (148 mg, 0.37 mmol), column CH2Cl2/MeOH 97/3, white foam, yield : 130 mg,
(88%); 1H NMR: (600 MHz, CDCl3) Mixture of conformers δ 8.95 (m, 1H), 7.94 (s, 1H), 4.23 (hept, J =
6.8 Hz, 1H), 4.16 (m, 2H), 3.85 (m, 2H), 3.70 (m, 2H), 3.61 (s, 2H), 3.56 (m, 2H), 1.49 (s, 9H), 1.33 (d, J
= 6.8 Hz, 6H); 13C NMR: (150 MHz, CDCl3) δ 164.7, 164.4, 157.2, 154.4, 151.1, 151.0, 126.5, 80.4, 47.3,
43.6 (broad), 42.6, 28.3, 27.9, 22.6; HRMS: (ESI-TOF) C18H28N6O4H+ expected: 393.2245. found:
393.2239.
tert-Butyl 3-((6-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-3-(hydrazinecarbonyl)pyridazin-4-
yl)methyl)-1H-indole-1-carboxylate (10b); Starting from 9b (164 mg, 0.28 mmol), column
1
CH2Cl2/MeOH 97/3, pale yellow foam, yield: 130 mg, (79%); H NMR (250 MHz, CDCl3) Mixture of
conformers δ: 9.13 (bs, 1H), 8.15 (bs, 1H), 7.61 (s, 1H), 7.50 (s, 1H), 7.33 (m, 2H), 7.18 (dd, J = 7.0, 7.8
13
Hz, 1H), 4.69 (s, 2H), 3.72 (m, 2H), 3.52 (m, 4H), 3.44 (m, 2H), 1.67 (s, 9H), 1.45 (s, 9H); C NMR
(150 MHz, CDCl3) δ: 164.4, 164.2, 157.2, 154.4, 150.7, 143.2, 129.7, 129.5, 125.1, 124.9, 124.8, 122.7,
118.8, 115.6, 115.5, 84.0, 80.4, 47.2, 42.5, 28.3, 28.2, 27.0; HRMS: (ESI-TOF) C29H37N7O6H+ expected:
580.2878, found: 580.2871.
(S)-tert-Butyl 4-(6-(hydrazinecarbonyl)-5-isobutylpyridazine-3-carbonyl)-2-isobutylpiperazine-1-
carboxylate (10c); Starting from 9c (219 mg, 0.47 mmol), column CH2Cl2/AcOEt 1/1, white foam, yield:
1
180 mg, (82%); H NMR (600 MHz, CDCl3) Mixture of conformers δ 9.03 (d, J = 9.6 Hz, 1H), 7.73 (s,
0.6H), 7.71 (s, 0.4H), 4.65 (d, J = 12.5 Hz, 0.4H), 4.59 (d, J = 13.0 Hz, 0.6H), 4.16-4.07 (m, 3H), 3.94 (d,
J = 13.2 Hz, 0.6H), 3.46 (dd, J = 13.6, 3.7 Hz, 0.4H), 3.20-2.94 (m, 4H), 2.00 (m, 1H), 1.60 (m, 1H), 1.53
13
(m, 1H), 1.47 (s, 5H), 1.46 (s, 4H), 1.20 (m, 1H), 0.96-0.92 (m, 9H), 0.83 (t, J = 6.8 Hz, 3H); C NMR
(150.9 MHz, CDCl3) δ 165.3, 165.0, 156.8, 156.7, 151.5, 144.4, 144.3, 131.0, 130.7, 80.4, 53.4, 50.1,
47.5, 45.7, 42.6, 40.8, 38.0, 29.5, 28.3, 24.7, 24.6, 22.8, 22.7, 22.6, 22.3; HRMS: (ESI-TOF)
C23H38N6O4H+ expected: 463.3027, found 463.3026.

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General procedure for the synthesis of the ureas :
To a solution of sodium nitrite (2 equivalents) in H2O (0.7 mL) was added at 0 °C a solution of HCl (1 M,
4 equivalents) and a solution of acetic acid (3 equivalents). After 5 min under vigorous stirring, a solution
of acyl hydrazide (1 equivalent) in THF (6 mL) was added dropwise. The mixture was stirred for another
20 min at 0 °C. Saturated aqueous Na2CO3 solution was then added until pH is basic, and the organic
phase is extracted with Et2O (3x10 mL). The organic fractions were collected, dried over Na2SO4 and
concentrated under reduced pressure to give the desired acyl azide as yellow oil. The azide was then
o
dissolved in anhydrous toluene (1 mL) and stirred for 1 h at 70 C to give the corresponding isocyanate.
The reaction mixture was cooled to rt and an amine (1.1 equivalent) was added. The resulting mixture
was stirred over night at rt, concentrated in vacuo and the residue purified by preparative TLC.
tert-Butyl 4-(6-(3-isopentylureido)-5-isopropylpyridazine-3-carbonyl)piperazine-1-carboxylate
(13a); Starting from 10a (55 mg, 0.14 mmol), Prep. TLC CH2Cl2/MeOH 97/3, white solid, yield: 34 mg,
1
(52%); H NMR: (250 MHz, CDCl3) Mixture of conformers δ 9.69 (m, 1H), 8.04 (s, 1H), 7.66 (s, 1H),
3.76 (m, 4H), 3.52 (m, 4H), 3.39 (m, 2H), 3.14 (hept, J = 6.7 Hz, 1H), 1.67 (m, 1H), 1.53 (m, 2H), 1.46
(m, 9H), 1.29 (d, J = 6.7 Hz, 6H), 0.93 (d, J = 6.5 Hz, 6H); 13C NMR: (62.5 MHz, CDCl3) δ 165.2, 154.6,
154.5, 154.2, 151.0, 136.8, 125.6, 80.3, 47.3, 42.6, 38.6, 28.3, 26.6, 25.9, 22.4, 21.2; HRMS: (ESI-TOF)
C23H38N6O4H+ expected: 463.3027. found: 463.3035.
tert-Butyl 4-(5-isopropyl-6-(3-(3-(methylthio)propyl)ureido)pyridazine-3-carbonyl)piperazine-1-
carboxylate (13b); Starting from 10a (55 mg, 0.14 mmol), Prep. TLC CH2Cl2/MeOH 97/3, white solid,
1
yield: 35 mg, (52%); H NMR: (250 MHz, CDCl3) Mixture of conformers δ 9.80 (m, 1H), 7.90 (s, 1H),
7.68 (s, 1H), 3.78 (m, 4H), 3.62-3.42 (m, 6H), 3.09 (hept, J = 6.7 Hz, 1H), 2.57 (m, 2H), 2.10 (s, 3H),
1.93 (m, 2H), 1.46 (s, 9H), 1.30 (d, J = 6.7 Hz, 6H); 13C NMR: (62.5 MHz, CDCl3) δ 165.1, 154.7, 154.5,
154.1, 151.2, 136.7, 125.8, 80.3, 47.3, 43.4 (broad), 42.6, 39.1, 31.5, 29.1, 28.3, 26.6, 21.2, 15.5; HRMS:
(ESI-TOF) C22H36N6O4SH+ expected: 481.2591. found: 481.2590.
tert-Butyl 3-((6-(4-(tert-butoxycarbonyl)piperazine-1-carbonyl)-3-(3-isopentylureido)pyridazin-4-
yl)methyl)-1H-indole-1-carboxylate (13c); Starting from 10b (47 mg, 0.08 mmol), Prep. TLC
1
CH2Cl2/MeOH 97/3, white solid, yield: 16 mg, (31%); H NMR: (250 MHz, CDCl3) Mixture of
conformers δ 9.63 (m, 1H), 8.15 (m, 1H), 8.04 (s, 1H), 7.49 (s, 1H), 7.42 (s, 1H), 7.39-7.15 (m, 3H), 4.06
(s, 2H), 3.70 (m, 4H), 3.47 (m, 4H), 3.37 (m, 2H), 1.68 (s, 9H), 1.61 (m, 1H), 1.51 (m, 2H), 1.46 (s, 9H),
0.87 (d, J = 6.4 Hz, 6H); 13C NMR: (62.5 MHz, CDCl3) δ 164.9, 154.8, 154.4, 151.0, 149.3, 135.6, 129.2,
128.9, 125.0, 122.8, 118.5, 115.6, 113.1, 84.2, 80.3, 47.3, 43.5 (broad), 42.5, 38.6, 28.3, 28.1, 25.9, 25.6,
22.4; HRMS: (ESI-TOF) C34H47N7O6H+ expected: 650.3660. found: 650.3654.
(S)-tert-Butyl 2-isobutyl-4-(5-isobutyl-6-(3-isopentylureido)pyridazine-3-carbonyl)piperazine-1-
carboxylate (13d); Starting from 10c (86 mg, 0.18 mmol), Prep. TLC Hexane/AcOEt 1/2, white solid,

HETEROCYCLES, Vol. 73, 2007
669
1
yield: 25 mg, (25%); H NMR: (600 MHz, CDCl3) Mixture of conformers δ 9.58 (m, 1H), 7.59 (s, 1H),
7.17 (d, J = 19.6 Hz, 1H), 4.62 (d, J = 12.5 Hz, 0.5H), 4.57 (d, J = 13.2 Hz, 0.5H), 3.98-4.35 (m, 3H),
2.91-3.48 (m, 5H), 2.48 (m, 2H), 2.03 (m, 1H), 1.69 (m, 1H), 1.61 (m, 0.6H), 1.54 (m, 2.4H), 1.48 (s,
13
4.5H), 1.47 (s, 4.5H), 1.30-1.42 (m, 2H), 0.93-1.00 (m, 15H), 0.87 (t, J = 5.8 Hz, 3H); C NMR: (150
MHz, CDCl3) δ 165.4, 154.9, 154.4, 154.2, 150.9, 150.8, 138.3, 129.8, 129.6, 80.3, 50.1, 47.5, 45.6, 42.8,
38.8, 38.7, 38.1, 28.4, 26.8, 26.0, 25.9, 24.8, 24.7, 22.9, 22.8, 22.6, 22.5, 22.4; HRMS: (ESI-TOF)
C28H48N6O4H+ expected: 533.3810. found: 533.3814.
General procedure for the Boc deprotection:
To a solution of the urea in CH2Cl2 (0.75 mL) at 0 oC was added TFA (0.25 mL). The mixture was stirred
for 3 h at rt and then concentrated in vacuo. The crude residue was purified on a short plug of silica if
necessary to afford the desired amines as trifluoroacetic acid salts. In the case of compound 1c iPr3SiH
was added for the deprotection of the indole.
1-Isopentyl-3-(4-isopropyl-6-(piperazine-1-carbonyl)pyridazin-3-yl)urea 2,2,2-trifluoroacetate (1a);
Starting from 13a (34 mg, 0.07 mmol), Column CH2Cl2/MeOH 9/1, white solid, yield : 34 mg, (97%); 1H
NMR: (600 MHz, CDCl3/CD3OD 95/5) Mixture of conformers δ 7.69 (s, 1H), 4.06 (m, 4H), 3.75 (m, 4H),
3.35 (m, 2H), 3.04 (hept, J = 6.7 Hz, 1H), 1.63 (hept, J = 6.7 Hz, 1H), 1.48 (m, 2H), 1.25 (d, J = 6.7 Hz,
13
6H), 0.89 (d, J = 6.6 Hz, 6H); C NMR: (150 MHz, CDCl3/CD3OD 95/5) δ 164.9, 154.6, 154.1, 150.1,
137.6, 125.9, 44.2, 43.5, 42.9, 39.4, 38.4, 38.3, 26.5, 25.7, 22.1, 20.8; HRMS: (ESI-TOF) C18H30N6O2H+
expected: 363.2503. found: 363.2448.
1-(4-Isopropyl-6-(piperazine-1-carbonyl)pyridazin-3-yl)-3-(3-(methylthio)propyl)urea 2,2,2-
trifluoroacetate (1b); Starting from 13b (35 mg, 0.07 mmol), Column CH2Cl2/MeOH 9/1, white solid,
1
yield : 34 mg, (94%); H NMR: (600 MHz, CDCl3/CD3OD 95/5) Mixture of conformers δ 7.71 (s, 1H),
4.07 (m, 4H), 3.60-3.40 (m, 6H), 3.04 (hept, J = 6.7 Hz, 1H), 2.54 (m, 2H), 2.07 (s, 3H), 1.90 (m, 2H),
1.27 (d, J = 6.7 Hz, 6H); 13C NMR: (150 MHz, CDCl3/CD3OD 95/5) δ 164.8, 154.8, 154.1, 150.2, 137.5,
126.0, 44.2, 43.5, 39.5, 38.9, 31.3, 28.8, 26.6, 20.8, 15.2; HRMS: (ESI-TOF) C17H28N6O2SH+ expected:
381.2067. found: 381.2052.
1-(4-((1H-Indol-3-yl)methyl)-6-(piperazine-1-carbonyl)pyridazin-3-yl)-3-isopentylurea (1c); Starting
from 13c (16 mg, 0.02 mmol), Column saturated solution of NH3 in CH2Cl2, white solid, yield : 8 mg,
1
(73%); H NMR: (600 MHz, CDCl3) Mixture of conformers δ 9.53 (m, 1H), 8.58 (s, 1H), 7.59 (s, 1H),
7.44 (s, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.24 (m, 1H), 7.13-7.09 (m, 2H), 4.09 (s,
2H), 3.79 (m, 2H), 3.72 (m, 2H), 3.40 (m, 2H), 2.99 (m, 2H), 2.91 (m, 2H), 1.67 (hept, J = 6.7 Hz, 1H),
1.53 (m, 2H), 0.93 (d, J = 6.6 Hz, 6H); 13C NMR: (150 MHz, CDCl3) δ 164.9, 154.7, 151.4, 136.6, 129.3,

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129.0, 126.5, 123.4, 122.8, 120.0, 118.3, 111.7, 108.0, 48.7, 46.4, 45.7, 43.6, 38.7, 38.6, 26.8, 25.9, 22.4;
HRMS: (ESI-TOF) C24H31N7O2H+ expected: 450.2612. found: 450.2610.
(S)-1-(4-Isobutyl-6-(3-isobutylpiperazine-1-carbonyl)pyridazin-3-yl)-3-isopentylurea (1d); Starting
from 13d (25 mg, 0.04 mmol), washed with saturated NaHCO3 solution, white solid, yield : 18 mg,
(90%); 1H NMR (600 MHz, CDCl3) Mixture of conformers δ 9.61 (m, 1H), 7.56 (s, 1H), 7.29 (d, J = 12.9
Hz, 1H), 4.61 (d, J = 12.3 Hz, 1H), 4.20 (m, 1H), 3.41 (m, 2H), 2.86-3.32 (m, 4H), 2.62 (m, 0.6H), 2.48
(d, J = 7.0 Hz, 2H), 2.30 (m, 0.4H), 2.04 (m, 1H), 1.77 (m, 0.4H), 1.68 (m, 1H), 1.62 (m, 0.6H), 1.53 (m,
2H), 1.35 (m, 1H), 0.88-0.99 (m, 18H); 13C NMR: (75 MHz, CDCl3) δ 164.8, 164.7, 154.8, 154.1, 150.9,
136.8, 129.7, 129.6, 54.0, 53.2, 47.8, 45.0, 42.8, 38.8, 38.7, 31.9, 30.3, 29.7, 29.4, 26.7, 26.0, 25.9, 24.3,
24.2, 22.7, 22.5, 22.4; HRMS: (ESI-TOF) C23H40N6O2H+ expected 433.3285. found 433.3277.
ACKNOWLEDGEMENTS
We thank Dr. D.-H. Huang and Dr. L. Pasternack for NMR spectroscopic assistance. We are grateful to
the ARCS Foundation (S.M.B.), the Spanish Ministerio de Educacion y Ciencia (E.M.), Novartis and the
Skaggs Institute for financial support.
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