Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors

Bioorganic & Medicinal Chemistry 12(2004) 1071–1089

Synthesis, structural investigations and biological evaluation of

novel hexahydropyridazine-1-carboximidamides, -carbothioamides

and -carbothioimidic acid esters as inducible nitric oxide synthase

inhibitors

Olaf Morgenstern,^a,* Heike Wanka,^bIlka Roser,^aAntje Steveling^band Beate Kuttler^b

^aErnst-Moritz-Arndt-Universitat Greifswald, Institut fur Pharmazie, Friedrich-Ludwig-Jahn- Straße 17,

D-17487 Greifswald, Germany

¨

^bErnst-Moritz-Arndt-Universitat Greifswald, Institut fur Physiologie, Greifswalder Straße 11a, D-17495 Karlsburg, Germany

¨

Received 22 July 2003; accepted 9 December 2003

Abstract—Local excess of nitric oxide (NO) has been implicated in b-cell damage, thus, a possible approach to the treatment of

autoimmune IDDM is the selective inhibition of inducible nitric oxide synthase (iNOS). A series of variously substituted hexahy-

dropyridazine-1-carbothioamides, -carbothioimidic acid esters and -carboximidamides was synthesized and dose-dependently eval-

uated as potential inhibitors of iNOS. The screening of the title compounds was performed with insulin-producing RIN-5AH cells

and a combination of IL1-1b and IFN-g as inducers of cellular NO production. The structure–activity analysis revealed that the

variation of substituents in the position 1 of the hexahydropyridazine strongly inﬂuences the inhibitory activity to iNOS as well as

being critical for RIN cell survival. Among the compounds tested, the hexahydropyridazine-1-carbothioamides showed particularly

signiﬁcant inhibitory eﬀects. However, for an eﬃcient iNOS inhibition substitution at the nitrogen of the 1-carbothioamide group is

important. Thus, the introduction of aliphatic chains such as propyl or butyl and of cyclic moieties such as cyclohexyl, 3-methoxy-

phenyl, and 4-methoxyphenyl (IC₅₀: 0.5–2.1 mM), respectively, provided compounds with similar inhibitory activity to aminogua-

nidine (IC₅₀: 0.3 mM), a common standard substance used for the selective inhibition of iNOS. However, the 1-carboximidamides,

which represent more structurally related semicyclic derivatives of aminoguanidine, caused only incomplete iNOS inhibition. The

hexahydropyridazine-1-carbothioimidic acid esters caused dose- and substituent-dependent damage of RIN-5AH cells. The toxicity

of the synthesized compounds increased markedly if aliphatic substituents at the exocyclic N atom(s) were replaced by variously

substituted aromatic rings.

1. Introduction

isms.⁴Besides these important functions, an over-

production of NO has been implicated in the

pathogenesis of various inﬂammatory and autoimmune

diseases.^5ꢀ8

The reactive metabolite nitric oxide (NO) is an intracel-

lular messenger that mediates several physiological

functions, including neurotransmission, vasodilatation,

modulation of leukocyte adhesion, antimicrobial and

antitumorial activities.^1ꢀ3NO is synthesized by a family

of three NO synthases (NOS). Whereas neuronal and

endothelial NOS are constitutively expressed (cNOS),

inducible NOS (iNOS) requires a stimulation by proin-

ﬂammatory cytokines or bacterial lipopolysaccharides

(LPS) and serves as defence enzyme against micoorgan-

In insulin-dependent type-1 diabetes (IDDM) the auto-

immune destruction of pancreatic b-cells is caused by an

inﬁltration of pancreatic islets by mononuclear leuco-

cytes. Autoreactive T-lymphocytes and a mixture of

cytokines released from diﬀerent mononuclear cells are

responsible for b-cell damage.⁹The cytokine inter-

leukin-1b (IL1b), alone or in combination with tumor

necrosis factor-a (TNFa) or interferon-g (IFNg ), induces

functional impairment and damage of rodent and

human islets in vitro.^10,11One mechanism of IL1

b-induced b-cell damage is due to an activation of b-cell

iNOS followed by an increased NO production.¹²NO

Keywords: Aminoguanidine; Hexahydropyridazine-1-carboximidamides;

Inducible nitric oxide synthase; Rat insulinoma cells (RIN).

* Corresponding author. Tel.: +49-3834-864848; fax: +49-3834-

864874; e-mail: omorgens@uni -g reifswald.de

doi:10.1016/j.bmc.2003.12.007

1072

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

also aﬀects the iron-containing respiratory chain

enzyme aconitase.¹³The consequence is a decrease in ATP

generation and subsequent cell death. The main producer

of IL1b are macrophages and dendritic cells, inﬁltrating

the pancreatic islets ﬁrst before autoreactive T-lympho-

cytes appear.¹⁴Therefore, an inhibition of NO produc-

tion may protect b-cells during an early phase of type-1

diabetes development.

2. Results and discussion

2.1. Syntheses and structural investigations

2.1.1. Synthesis of hexahydropyridazine. In order to

achieve the synthesis of the title compounds 4a–v

(Scheme 1, Table 2), it should be possible to start

from hexahydropyridazine 1 (piperidazine; N,N⁰-tetra-

methylene hydrazine).³²Hexahydropyridazine represents

an important starting material for diﬀerent herbicids and

fungicids with [1,2,4]triazolo[1,2-a]-^33ꢀ36or [1,3,4]thia-

diazolo[3,4-a]pyridazine structure.^36ꢀ45The classical

preparation method published by Alder et al.³²starts

with the addition of buta-1,3-diene to azodicarboxylic

acid dimethyl ester and has frequently been mod-

iﬁed^{40,46-49,54,55}or directly utilized by several authors.^50ꢀ53

Further patented⁵⁶and modiﬁed^57,58routes to 1 begin

with the potassium salt of diisobutyric hydrazine,⁵⁹with

hydrazine-1,2-dicarboxylic acid esters,⁶⁰with 1-amino-

pyrrolidine,⁶¹with butane-1,4-diamine,⁶²and with 2,2-

disubstituted hexahydropyrazolo[1,2-a]pyridazine-1,3-

diones,⁶³respectively. Up until now, only Marquis⁶⁴

reported the hydrogenation of pyridazine (with metallic

sodium in ethanolic solution), but instead of the form-

ation of 1 a ring cleavage occurs under formation of

butan-1,4-diamine.

However, for therapeutic use only selective inhibitors of

iNOS are suitable because NO generated by cNOS is

necessary for the maintenance of blood glucose concen-

tration by regulation of both insulin and glucagon

secretion.^15,16A selective inhibitor of iNOS is amino-

guanidine (hydrazine carboximidamide, AG). AG eﬀec-

tively reduces the cytokine-induced NO production of

pancreatic islets and rat insulinoma cell line (RIN

cells)^17,18and reverses IL1b inhibited insulin release in

vitro.¹⁹In transgeneic mice overexpressing iNOS in the

pancreatic islets, it was reported that all animals devel-

oped diabetes within an age of 4 weeks. Treatment with

AG prevented or delayed the onset of diabetes.²⁰

Administration of AG to young diabetes prone BB rats

or NOD mice also delayed diabetes onset.^21,22However,

there are side eﬀects that raise doubt about the use of

AG as therapeutic drug. Thus, application of AG to

wild type and iNOS knockout rats led to weight loss

and rendered them more susceptible to infections.²³

Furthermore, rats injected intravenously with AG (1–50

mg/kg body weight) showed a decreased pancreatic

blood ﬂow.²⁴In streptozotocin diabetic mice, adminis-

tration of AG (50 mg/kg body weight) failed not only to

prevent development of diabetes and insulitis but AG

treated mice also showed an increased mortality com-

pared to control mice treated with streptozotocin plus

saline.²⁵Finally, the selectivity of AG towards the iNOS

isoform is only achieved at low inhibitor concentrations;

higher AG doses inhibit all three NOS-isoforms, that is,

inhibition of cNOS also takes place.^26,27

To achieve a more ready access to 1 in relation to the

above mentioned methods, we again investigated the direct

hydrogenation of commercially available pyridazine

(hydrogen atmosphere, Adams catalyst, 5 bar; 25 ^ꢁC)

with the desired result. Because of the oxidation-sensi-

tivity of the batches, immediately after hydrogenation

the free hexahydropyridazine base was saliﬁed in ethe-

ric-ethanolic solution with dry hydrogen chloride gas to

give the monohydrochloride⁴⁶in good yields. To suppress

the formation of mixtures with the dihydrochloride³²

strict control of the hydrogen ion concentration is

required.

a

The modiﬁcation of the AG’s structure might yield

analogues with potent iNOS inhibitory activity but with

less side eﬀects. Moreover, it has been shown that the

amidine partial structure is an eﬀective iNOS inhibitory

pharmacophore.^28,29Open chained as well as cyclic

amidines were identiﬁed as inhibitors of both cNOS

and/or iNOS. In neuroinﬂammatory diseases pyrid-

azine-based compounds were also used as inhibitors of

iNOS.^30,31

Here we report a series of hexahydropyridazine-1-car-

boximidamides representing novel semicyclic amino-

guanidine derivatives with various stereochemical,

electronical, and bonding properties. In relation to the

known NOS inhibitory eﬀects of S-alkyl isothioureas,

for example, S-ethyl isothiourea (SEITU), the biological

activities of the related isosters such as the hexahy-

dropyridazine-1-carbothioimidic acid esters and the

carbothioamides, respectively, were also of interest. All

three types of hexahydropyridazine derivatives have

been evaluated for concentration dependent inhibitory

activity to cytokine-induced iNOS in insulin producing

RIN-5AH cells as well as toxicity to these cells.

Scheme 1. Reagents and conditions: (i) 2a: KSCN, H₂O, ꢀ; 2b–q:

TEA, related R¹-N¼C¼S, CH₂Cl₂, room temperature; (ii) CH₃I,

CH₂Cl₂, room temperature; (iii) NH₃or related R²R³NH, methods

A—E (see Experimental).

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1073

2.1.2. Syntheses of hexahydropyridazine-1-carbothio-

amides and hexahydropyridazine-1-carbothioimidic acid

methyl esters. The subsequent preparation of the ﬁrst

members of the hitherto unknown title compounds 4 was

planned in analogy to the pyrazolidine-1-carboximid-

amides. Their synthesis should be done as already

reported⁶⁵without isolation of intermediates, utilizing 1

and diﬀerent N-aryl and N,N⁰-diaryl-S-methyl isothio-

uronium iodides, respectively, as reactants (Scheme 1).

Table 1. 1,2,3,4,5,6-Hexahydropyridazine-1-carbothioamides 2 (X¼S,

Y¼NHR¹) and 1,2,3,4,5,6-hexahydropyridazine-1-carbothioimidic

acid methyl ester hydroiodides 3 (X¼NH⁺R¹I^ꢀ, Y¼SCH₃)

2,3

R¹

k⁰^a

a

b

c

H

CH₃

C₂H₅

C₃H₇

0.36

0.73

1.08

1.55

1.34

1.57

2.27

2.74

3.22

2.05

2.76

2.37

2.60

1.64

3.02

3.03

3.50

With the S-methyl-N,N⁰-diphenyl isothiouronium

iodide, which is frequently a reacting partner in the

reaction with pyrazolidine,⁶⁵considerable diﬀerences

between the reactivities of 1 and the ﬁve membered

homologue pyrazolidine, respectively, were observed.

These diﬀerences in the reactivities of both heterocycles

are evidently caused by their diﬀerent stereochemical

properties.^66,67In addition, even with a varity of other

substituted S-methylisothiouronium salts and by mod-

ifying the reaction conditions all attempts failed to

transform the hexahydropyridazine 1. For this reason,

the synthesis of S-(4-nitrobenzoyl)-1,3-diphenyl 5 iso-

thiourea from 1,3-diphenylthiourea and 4-nitrobenzoyl

chloride and its reaction with 1 was attempted. How-

ever, instead of the desired 5, the related, instable 1-acyl

thiourea 6⁶⁸was isolated. Interestingly, in the reaction

with 1 this compound acts as a donor of phenylisothio-

cyanate under formation of N-phenylcarbothioamide 2j

(Table 1) and 4-nitrobenzanilide.⁷³

d^b

e^b

f

CH₂¼CH–CH₂

(CH₃)₂CH

C₄H₉

C₆H₅CH₂CH₂

Cyclohexyl

C₆H₅

3-CH₃–C₆H₄

4-CH₃–C₆H₄

2-CH₃O–C₆H₄

4-CH₃O–C₆H₄

4-NO_2--C₆H₄

Naphth-1-yl

Naphth-2-yl

g^b

h

i

j

k

l

m

n

o^c

p

q

^aHPLC capacity factor for compounds 2, HPLC conditions: see

Experimental.

^bNon-crystalline mass with compounds 3.

^cIsolation of compound 3o failed.

careful done evaporation of an aqueous solution of

equimolar amounts of 1 hydrochloride and potassium

thiocyanate.

The combinatorial synthesis of 2b–q was also attemp-

ted. Thus, appropriate conditions for the high perfor-

mance liquid chromatography (HPLC) separation were

developed (HPLC conditions see Experimental, k⁰

values are listed in Table 1). In summary, the combina-

torial synthesis of compounds 2 was accompanied by

undesired and not further characterized by-products;

the rates of the 2-formation were lower than 100%

(exceptionally 2b,e,m,j). These rates decrease at the

same time when the reactivity of the isothiocyanate

decreases; this point speaks for the enforced participa-

tion of the isothiocyanates selectively reacting in the

formation of by-products.

These ﬁndings lead us to attempt a new route (Scheme

1, steps i–iii) to the title compounds 4 via the hexahy-

dropyridazine-1-carbothioamides 2a–q (Table 1) and

hexahydropyridazine-1-carbothioimidic acid methyl

ester hydroiodides 3a–q (Table 1), which was realized as

reported in the past.⁶⁵Whereas the 3 and the corres-

ponding free bases are hitherto unknown, in various

patents^{34,37ꢀ42,44,45}several compounds with the general

structure 2 are utilized as intermediates for heterocyclic

crop protection chemicals. With a few exceptions,³⁴

these products represent polysubstituted and also halo-

genated N-phenyl derivatives. Various hexahydropyr-

idazine-1-carbothioamides (ref 34: 2f, i, l, n) also

prepeared in our work, are explicitly named as starting

products for [1,2,4]triazolo[1,2-a]pyridazines, but only

very few of these have been directly characterized (ref

33: 2j, l).

Both with regard to the expected problems due to the

diﬀerent behavoir of the reactants and in order to an

extensive characterization of the hitherto unknown

hexahydropyridazine derivatives 3 and 4 the parallel

synthesis approach was utilized in the preparation of

these series of compounds.

The preparation of the hexahydropyridazine-1-carbo-

thioimidic acid methyl ester hydroiodides 3a–q pro-

ceeded under mild conditions by reacting iodomethane

with the carbothioamides 2 (Scheme 1, step ii), where

the N-alkyl substances took the longest reaction times.

However, the preparation of compound 3o from 2o was

not achieved even when heating with dimethyl sulfate.

The isolation of the compounds 3 was complicated

by poor tendency of most of these compounds to

crystallize.

In principle, the compounds 2b–q may be prepared from

hexahydropyridazine (1) hydrochloride and related iso-

thiocyanates (Scheme 1, step i) in the presence of a base

in an aprotic solvent in good yields. They crystallize out

very nicely and show, in contrast to pyrazolidine,⁷⁰only

a minor tendency to form 1,2-dicarbothioamide deriv-

atives. The synthesis of 2a proceeded in good yields by

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O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

2.1.3. Synthesis of hexahydropyridazine-1-carboximid-

amides. The synthesis of the compounds 4a–v (Table 2)

proceeded under mild conditions by allowing either

ammonia or amines to react with the hydroiodides 3

(Scheme 1, step iii). Thus, 4a, which embodies as the

N¹,N²-tetramethylene derivative of aminoguanidine the

parent compound of the 4, was synthesized by reacting

3a with gaseous ammonia.

the applied 3 and the alkaline hydrolysis of the formed 4

are suppressed. The latter fact also becomes very clear

in the preparation of the N-aryl derivatives 4i–k.

Methylamine was used with an excess in a methanol-

aqueous mixture; the undesired side reactions were

attenuated because of the stronger nucleophility of the

methylamine compared to ammonia. However, only

moderate yields of the aromatic substituted derivatives

4r,s were achieved.

Because of the varying reactivity of the 3 and the amines

utilized, monitoring of the reaction batches by TLC was

done. As a result, the application of the weak nucleo-

philic aromatic amines was found to be unpractically,

and for this reason the synthesis of N¹,N²-diaryl sub-

stituted derivatives 4 is not reported here. However, the

monoaryl substituted compounds 4i–k and 4q–t were

obtained from the related N-aryl derivatives 3. With the

exception of the most of the aryl derivatives the isolated

compounds 4 are mostly low melting substances that

crystallized poorly (some are hygroscopic compounds;

especially 4b). In order to crystallize the title com-

pounds, the products were dried and covered with die-

thylether for several days.

All attempts to synthesize some N¹,N¹,N²-trisubstituted

derivatives 4 in an analogous manner with dimethyl-

amine failed. Most likely of this was because of stronger

basicity in relation to methylamine (competing reac-

tions, isolation of dimethylamine hydroiodide), and so

these compounds are not reported here.

That the N¹,N¹-disubstituted and the N¹,N¹,N²-trisub-

stituted compounds 4 can be prepared at all, was

demonstrated with 4u,v. These N¹,N¹-tetramethylene

derivatives were obtained by reaction of 3a and its N-

methyl analogue 3b with the secondary amine pyrroli-

dine, respectively. However, the successful preparation

of further N¹,N¹-disubstituted and the N¹,N¹,N²-trisub-

stituted derivatives 4 requires additional investigations

with regard to suﬃcient reaction conditions, and this

will be reported in a later paper.

The investigations on the reaction conditions led to

variations of the synthesis of compounds 4a–v (methods

A–E, see Experimental). If possible, methods A and B

utilizing ammonia should be favoured because of the

high volatility of the ammonia the working-up is sim-

pliﬁed. The advantage of a non-aqueous reaction med-

ium makes clear the transformation of 3h to 4h by

reaction with ammonia (yield 87% versus 30%); the

competing reactions consisting of the saponiﬁcation of

2.1.4. Structural investigations. All the series of com-

pounds 2, 3, and 4 described in the experimental part

were analyzed by TLC and found to be chemically uni-

form. The data resulting from the elemental analysis

were in accordance with the calculated values within the

usual range of tolerances (ꢂ0.4% of the calculated

values for C, H, N). Compared with hexahydropyr-

idazine 1, the N-substituted bases 2–4 are much less

oxygen sensitive. There was no evidence in the NMR

nor in the mass spectra for the formation of an unsatu-

rated heterocyclic ring. The chemical shifts of the

proton and carbon NMR signals for the hexahydro-

pyridazine system of the synthesized 2–4 are in good

agreement with the data given by other authors.^52,53

However, caused of the oxidation of iodide to iodine,

3 and 4 tended to form mixtures consisting of the

hydroiodide and the related free base during long-term

storage in the air.

Table 2. 1,2,3,4,5,6-Hexahydropyridazine-1-carboximid-amide hydro-

iodides 4^a

4

R¹

R²

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

H

CH₃

C₂H₅

C₃H₇

NH₂

CH₂–CH¼CH₂

H

C₄H₉

NHCH(CH₃)₂

NH₂

The proton NMR spectra of the hexahydropyridazine-

1-carbothioamides 2 show no evidence for the forma-

tion of 1,2-dicarbothioamides side products. Due to

coupling with the C(3)H₂structure of the heterocycle,

the N(2)H signals appear as triplets (in the spectrum of

2i as broad singulet). Moreover, the spectra did not

provide any indication of the tautomeric carbothioimide

structure. Thus, the proton NMR spectrum of 2a shows

the existence of two equivalent protons of the unsub-

stituted thiocarbamoyl group. In the case of the N-alkyl

derivatives 2b,c–h the signals from the N-alkyl sub-

stituents show splitting patterns, that are consistent with

the assigned carbothioamide structure. The comparison

of the carbon NMR spectra of the 2 with those of the

corresponding 3 provides evidence of the favoured

CH₂–CH₂C₆H₅

C₆H₅

2-CH₃O–C₆H₄

4-CH₃O–C₆H₄

CH₃

NH₂

NHCH₃

NHC₂H₅

NHC₃H₇

NHC₄H₉

NHCH₂CH₂C₆H₅

NHCH₃

Pyrrolidino

CH₃

C₆H₅

4-CH₃–C₆H₄

2-CH₃O–C₆H₄

4-CH₃O–C₆H₄

H

s

t

u

v

CH₃

^aThe given structures for the 4 are derived from the NMR spectra of

the compounds (conditions: see Experimental).

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1075

existence of 2a–q in the CS-NH form; the positions of

the signal of the only sp²hybridized carbon atom of the

compounds 2a–d,f,g,i is clearly situated at lower ﬁeld

(about 180 ppm) than in the spectra of the analogous 3

(about 167 ppm). Similar results were obtained for the

other isolated carbothioamides 2.

ratios. Moreover, the proton resonance spectra allow an

assignment as to which of the substituents are linked to

each of the diﬀerent hybridized nitrogen atoms N¹(sp³-

hybridization) and N²(sp²-hybridization) of the exo-

cyclic amidino group. Assigning the farthest low ﬁeld

shifted signals of the exchangable protons to the posi-

tive charged nitrogen atom and simultaneously taking

into account the integrals and the splitting patterns

(coupling of the nitrogen linked protons with the a car-

bon protons of the alkyl substituents) of the signals of

all the exchangable protons, the structures of the cations

are clearly apparant (Table 2). In addition, no evidence

was found in the proton spectra which might point to

the simultaneously presence of further synionic

structures.

The fragmentation behavior of the molecular ions of 2,

formed by electron impact in mass spectrometry, is

mainly characterized by a retroaddition yielding the

related isothiocyanates and hexahydropyridazine. This

was especially the case with the N-alkyl derivatives 2,

which gave intensive peaks at m/z 86 (M^+.–R–NCS).

Furthermore, the mass spectra did not provide any

indications of the elimination of HS^.or H₂S, which also

speaks for the existance of the carbothioamide form.

The ¹³C NMR peaks of the sp²-hybridized carbon

atoms of the amidino group appear between 154.32

ppm (4f) and 158.05 ppm (4k) for the N²-unsubstituted

carboximidamides, in a small range from 158.31 ppm

(4p) to 158.92ppm ( 4l) for the N¹,N²-dialkylated com-

pounds 4, and again at a little higher ﬁeld between

155.29 ppm (4q) and 157.92( 4t) for the N²-aryl-N¹-

methyl derivatives 4q–t. In relation to those of the car-

bon atoms of the carbothioimidic acid ester structure of

the 3 (see above), these are clearly shifted to higher ﬁeld.

In order to deﬁnitely assign the peaks of the carbon

NMR spectra of the compounds 4n–o,r,t, the corres-

ponding DEPT 135 spectra were also analyzed.

The proton NMR spectra of the hexahydropyridazine-

1-carbothioimidic acid methyl ester hydroiodides 3 gave

no evidences for an alternative alkylation of the N(2) of

the hexahydropyridazine ring. Normally, the signals of

the N(2) partial structure appear in the range from 5.50

ppm (3b) to 6.23 ppm (3m), but in the case of the

naphthyl derivatives 3p,q the related signal was not

observed. However, the carbon NMR spectra of all the

compounds 3 argue for S-alkylation of the carbothio-

amide structure of the 2. Assuming the N-methylation

of the carbothioamide structure, the position of the sig-

nals of the C¼S group related to the analogous 2 should

not be shifted to such high ﬁelds. Moreover, the loss of

methanethiol from the molecular ion of 3a in the mass

spectrometry and the evolution of this compound in the

reaction of 3 with amines (subsequent transformation to

the 4, step iii, Scheme 1) prove the S-methylation of the

carbothioamide structure.

The electron impact mass spectra of the hexahydropyr-

idazine-1-carboximidamide hydroiodides 4 gave the free

bases and are in accordance with the structures

assigned. The intensities of the peaks of the molecular

ions are greatest with N¹-unsubstituted 4a–k, in which

the molecular ions of the arylated derivatives 4i–k are

additionally stabilized by the aromatic system. The mole-

cular ion peaks of the N¹,N²-dialkylated 4 are char-

acterized by intensities of only ca. 10% of the base

peaks (normally at m/z 86). Again, compared with the

N¹,N²-dialkylated products, the N²-aryl-N¹-methyl

derivatives 4q–t yield more intensive molecular ion

peaks (17–41%). The most favoured initial fragmenta-

tion of the molecular ions of the bases 4 constists of a

retroadditon, which obviously proceeds under forma-

tion of the neutral, related carbodiimides and the hexa-

hydropyridazine cation radical (m/z 86). Exceptions to

this were encountered with 4s (resulting from the ortho

located methoxy substituent) and the N¹,N¹-tetra-

methylene compounds 4u,v. The latter, structurally

related compounds, diﬀer considerably in the fragmen-

tation behavior of the molecular ions. However, both

mass spectra of 4u,v indicate the elimination of pyrroli-

dine from the molecular ion [4u: m/z 71 (75%); 4v: m/z

30 (38%)]. In contrast, all the other mass spectra of the

4-series did not give evidence for peaks arising form the

amine component, which is generated from the carbox-

imidamide structure.

With all the investigated carbothioimidic acid esters 3,

two types of exchangable protons exist in a 1:1 ratio

(with 3a 2:1). Because of the energetically favoured for-

mation of mesomeric cations, the protonation of the

carbothioimidic acid ester structure should occur at

the nitrogen atom, and this was supported by the clear

coupling with the a proton of the N-(cyclo)alkyl group

in the spectra of 3f and 3i. With all the other com-

pounds 3 the NH peaks for the –C(SCH₃)¼NH^ꢃ-struc-

ture appear as broad singulets between 8.29 ppm (3a)

and 10.18 ppm (3i).

With the hexahydropyridazine-1-carboximidamide

hydroiodides 4 the protonation site and the tautomer-

ism involving the amidino group are of particular

interest. The proton NMR spectra of the compounds

4a–v clearly indicate that the amidino group is the

site of protonation due to the formation of resonance-

stabilized cations. No evidence was found that

suggest protonation of the nitrogen atoms of the

hexahydropyridazine ring. Thus, compounds 4a,u,v

possess two types of exchangeable protons [integrals

ratio (amidino group protons : N(2)H atom): 4a: 4:1;

4u: 2:1; 4v: 1:1]. The other compounds 4b–t are char-

acterized by three types of magnetically non-equivalent

and exchangeable protons; these derivatives conse-

quently have diﬀerent, substitution dependent integrals

2.2. Biological evaluation

The screening of the title compounds for iNOS inhibi-

tion was performed with insulin-producing RIN-5AH

1076

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

cells (rat insulinoma cell line) and a combination of IL1-

b and IFN-g to induce NO production. Their usefulness

was determined by the exclusion of compound-mediated

cell death and by their inhibitory potency to iNOS. If

there was a complete inhibition of NO production the

eﬃciency of compounds was quantiﬁed by the IC₅₀

value.

2.2.1. Evaluation of hexahydropyridazine-1-carbothio-

amides (HPC). The dose-dependent eﬀects of the

derivatives on cytokine-induced NO production in com-

parison with aminoguanidine (AG) are shown in Figure

1. AG caused a dose-dependent reduction of cytokine-

induced NO-production. The inhibitory activity of AG

on iNOS was characterized by an IC₅₀value of 0.3 mM.

Figure 1. Inhibition of cytokine-induced iNOS by aminoguanidine and several hexahydropyridazine-1-carbothioamides (HPC) 2 depending on the

concentration and the substitution of the carbothioamide group, respectively. Inﬂuence of HPC derivatives on cytokine-induced NO production: (a)

and (b) exocyclic N-substitution with aliphatic chains, (c)–(e) exocyclic N-substitution with cyclic moieties. Curves were obtained by ﬁtted nonlinear

regression analysis using a sigmoidal dose response. Simultaneously to the determination of NO production using the Griess reaction the inﬂuence of

inhibitors on RIN-5AH cell viability was tested to calculate a potential toxic eﬀect of derivatives.

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1077

The hexahydropyridazines possessing a substituted 1-

carbothioamide structure led to compounds with iNOS

inhibitory properties, but compared to AG with reduced

potency as expressed by the degree of inhibition and the

enhanced IC₅₀values (Table 3). 5 out of 16 compounds

(2a, b, j, o, q) showed only an incomplete inhibitory

activity at the concentrations investigated. The N-sub-

stitution of the carbothioamide group by aliphatic

residues brought about complete inhibition (Fig. 1a).

The IC₅₀values decreased continuously from 2.1, 1.1 to

0.83 mM as a function of the chain length for the com-

pounds N-ethyl, N-propyl, and N-butyl-HPC (2c,d,g).

However, N-butyl-HPC 2g showed also toxic side eﬀects

on RIN cells at a concentration of 5.0 mM. The

exchange of the unsaturated allyl (2e, IC₅₀: 2.7 mM) for

the (iso)propyl residue still increases further the inhibitory

potency (2d, IC₅₀: 1.1 mM, 2f, IC₅₀: 1.2mM) ( Fig. 1b).

the used concentrations. Toxic eﬀects on RIN cells were

detectable with N-(4-methoxyphenyl)-(2n) and N-cyclo-

hexyl-HPC (2i) at 5.0 mM (Table 3).

Extension of the aromatic system (naphthyl derivatives)

led to the compounds with both complete (2p, IC₅₀: 1.2

mM) and incomplete inhibitory activity (2q) (Fig. 1e).

These results indicate that besides compounds contain-

ing the amidine function like aminoguanidine also some

hexahydropyridazine derivatives 2 possessing a semi-

cyclic thiosemicarbazide partial structure are able to

inhibit iNOS, however, with a less activity (IC₅₀values

of 0.6–2.7 mM).

2.2.2. Evaluation of hexahydropyridazine-1-carbothioimi-

dic methyl esters. With one exception (3b) the S-methy-

lation of the pyridazine-1-carbothioamide derivatives

caused a dramatic increase in toxicity (Table 3). In gen-

eral, the compounds bearing cyclic residues at the exo-

cyclic N atom (3i–q) were more toxic than the aliphatic

substituted derivatives (3a–f). Compound 3b showed no

toxicity at the used concentrations and was able to

inhibit NO production completely with an IC₅₀value of

1.2mM (data not shown). The results reveal that the

exchange of the carbothioamide structure for the car-

bothioimidic acid methyl ester group is possibly crucial

for the activities of compounds and therefore for the

survival of RIN-5AH cells.

The evaluation of such 2-derivatives, which are char-

acterized by a cyclic moiety, showed that cyclohexyl-

HPC 2i was a more eﬀective compound (IC₅₀: 0.5 mM)

than the aromatic phenyl-HPC 2j and N-(2-phenyl-

ethyl)-HPC 2h (non active and IC₅₀: 2.7 mM, respec-

tively) (Fig. 1c). The insertion of a chain between the

hexahydropyridazine ring and the phenyl ring not only

increased the inhibitory potency but also enhanced the

toxic behaviour (Fig. 1d). The inhibitory properties

were improved depending on the substituent at the

phenyl ring as follows: 4-nitro (2o)<2-methoxy

(2m)<4-methoxy (2n)<3-methyl (2k) (IC₅₀: 2.3, 2.1, 0.9

and 0.6 mM, respectively). According to these results

the most eﬀective compound in these series was 2k with

an IC₅₀of 0.6 mM and without any toxic side eﬀects at

2.2.3. Evaluation of hexahydropyridazine-1-carboximida-

mides (HPCI). The degrees of inhibition of cytokine

induced NO production by HPCI derivatives and their

toxic dose to RIN-5AH cells are demonstrated in Table 3.

Table 3. Toxic eﬀects of hexahydropyridazine-1-carbothioamides (compounds 2),-carbothioimidic acid methyl ester hydroiodides (compounds 3),

and-carboximidamide hydroiodides (compounds 4) on RIN-5AH cells and inhibition of inducible NO-synthase activity by 2 and 4

2

TC (mM)^a

Inhibition (%)^b

IC₅₀(mM)

3

TC (mM)^a

4

TC (mM)^a

Inhibition (%)^b

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

n.t.

5.0

n.t.

5.0

n.t.

no data

n.t.

5.0

n.t.

69.8

58.6

87.0

92.6

n.d.

2.1

1.1

2.7

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

2.5

n.t.

2.5

no data

2.5

no data

2.5

0.5

0.25

0.1

0.25

0.1

no data

2.5

1.0

a

b

c

d

e

f

g

h

i

j

k

l

m

n

o

p

q

r

n.t.

5.0

n.t.

no data

2.5

n.t.

no data

2.5

61.7

61.2

32.0

46.1

44.6

43.9

78.4

no data

26.9

23.8

22.9

32.2

33.6

49.1

no data

47.2

26.9

31.9

28.4

23.5

35.0

24.8

82.6

88.21.2

88.8

88.6

83.3

30.3

0.8

2.7

0.5

n.d.

0.6

no data

96.8

no data

90.22

90.5

33.3

87.1

.1

0.9

n.d.

1.2

59.0

n.d.

2.5

n.t.

s

t

u

v

n.t.

^aToxic concentration (expressed in mM) of synthesized compounds to RIN-5AH cells measured by trypanblue exclusion test. At the given drug

concentration all RIN cells were trypanblue positive; n.t.=not toxic up to a concentration of 5.0 mM.

^bDegree of inhibition (%) of cytokine-induced NO production by a concentration of 5 mM or at the next lower concentration studied. IC₅₀values

were only determined if the degree of iNOS inhibition was greater than 80%. All data based on the mean value of at least ﬁve to six independent

experiments; n.d.=not determined. No data=compounds could not be dissolved completely.

1078

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

Although the 1-substituent of the hexahydropyridazine

ring represents an amidino group [–C(¼N–R¹)NR²R³]

and these compounds are structurally close to the lead

AG, the inhibitory activities were dose-dependent but

incomplete. The most active compounds were the

unsubstituted parent compound (4a) of the HPCI 4 and

the monosubstituted derivatives possessing the smallest

(4b) and the largest (4g) aliphatic chain. Additional

substitution, as found in the N¹,N²-disubstituted (4l–p)

and tetramethyl-HPCI derivatives (4u, 4v), caused a

further loss of inhibitory activity. In relation to the 1-

carbothioamides 2, the HPCI derivatives 4 probably may

not ﬁt eﬃciently into the active site of iNOS. Another

reason may be that the 4 underlie a lag in cell uptake.

4. Experimental

4.1. Chemistry

4.1.1. General. The reported melting temperatures were

determined on a Koﬂer-Boetius apparatus type PHMK

78/1910 (VEB Analytik Dresden) and are uncorrected.

Elemental analyses were done with the 2400 CHN Ele-

mental Analyzer (Perkin Elmer) and were in accordance

with the calculated values within the usual range (0.4%)

of tolerances. The spectra were recorded with the follo-

wig instruments and conditions: IR spectra: FT-IR 1600

(Perkin–Elmer); H and ¹³C NMR spectra: AVANCE

1

DPX 200 and ARX 300 (¹H NMR of 3p,q and ¹³C

NMR of 4t only) (both from Bruker Analytische

Meßtechnik GmbH); room temperature, internal stan-

dard tetramethylsilane; mass spectra: M 40 AMD

(Intectra GmbH), electron impact, energy 70 eV, (only

peaks>10% are listed). The gas chromatographic

investigations were done with a FISON Instruments GC

8065 coupled with a mass spectrometric detector MD

800 (70 eV); column: Macherey-Nagel MN FS Hydro-

dex b-PM (25 mꢄ0.25 mm); column prepressure: 10 psi

(70 kP); column temperature 140 ^ꢁC. The HPLC inves-

tigations were done with a LaChrom system (Merck

Hitachi) consisting of pump L-7100, autosampler

L-7200, column thermostat L-7350, solvent degasser

L-7612, interface D-7000, and diode array detector L-

7450 (further data are given below). For TLC aluminia

foil covered with Kieselgel GF₂₅₄(Merck) was utilized

as stationary phase, and as mobile phases the following

mixtures were employed: n-hexane/acetic acid ethyl

ester 1:1 (v/v) for the compounds 2, and n-hexane/acetic

acid ethyl ester/triethyl amine 6:3:1 (v/v/v) for the deri-

vatives of 3 and 4. The substances were detected with

UV radiation (l=254 nm) or Munier spray reagent⁷¹

(all the compounds) and iodine azide reagent by Awe⁷²

(2 and 3). All the members of the series of title com-

pounds 2–4 characterized in the experimental part were

obtained by parallel synthesis approach.

HPCI derivatives containing aromatic residues (4i–k, p–t)

were toxic to RIN-5AH cells (Table 3). Therefore, the

observed dose-dependent reduction of nitrite accumula-

tion is due to the death of RIN cells and not to an

inhibitory eﬀect to iNOS.

3. Conclusions

A number of hexahydropyridazine-1-carbothioamides,

-carboximidamides and -carbothioimidic acid methyl

esters have been prepared and tested with regard to

their in vitro inhibition of cytokine-induced NO pro-

duction by RIN-5AH cells. Especially the substituent in

the position 1 of the hexahydropyridazine derivatives

was critical for their usefulness as inhibitor. Whereas

most hexahydropyridazines with a 1-carbothioamide

group (compounds 2) caused a complete inhibition of

NO production the compounds lost their activity com-

pletely after S-methylation forming hexahydropyridazine-

1-carbothioimidic acid methyl ester (compounds 3). The

substitution by a carboximidamide group (compounds

4) also resulted in a decrease of inhibitory activity which

was characterized by an incomplete reduction of NO.

This eﬀect may be due to a structural mismatch between

the active site of iNOS and the inhibitor or possibly to

an ineﬀective uptake by the cells. Thus, only some few

derivatives of hexahydropyridazine-1-carbothioamides

particularly compound 2k were identiﬁed as potential

iNOS inhibitors. These compounds are taken up by the

RIN cells and are suﬃciently stable and eﬃcient to exert

their inhibitory activity in intact cells. Therefore, pyrid-

azine-based derivatives are not only able to inhibit

iNOS of neuronal cells^30,31but also of pancreatic insu-

lin-producing cells. However, before they should be

used in vivo the compounds have to be tested with

regard to their selectivity against iNOS and cNOS. First

hints concerning the isoform selectivity are given by the

spontaneous cNOS mediated NO production of RIN

cells. A concentration of 5 mM aminoguanidine caused

a decrease of spontaneous NO production and therefore

a possible inhibition of cNOS whereas 2k did not show

this eﬀect (data not shown). In case of a high iNOS

speciﬁcity of 2k this substance could be tested for their

eﬀect to isolated islets in vitro and could be valuable for

the treatment of diabetic syndrome because low amounts

of NO are still physiologically necessary for hormone

release by pancreatic islet cells.

4.1.2. 1,2,3,4,5,6-Hexahydropyridazine hydrochloride

.

(1 HCl). A mixture of 0.138 mol (11.04 g) pyridazine in

30 mL of absolute C₂H₅OH and 2.0 g Adams catalyst

(PtO₂ꢄx H₂O, 80% Pt) was shaken vigourously in a

hydrogen atmosphere (initial pressure 5 bar) at 25 ^ꢁC

for 6 h. Subsequently, the catalyst was decanted oﬀ, the

reaction vessel was rinsed with 20 mL of absolute

C₂H₅OH, the remaining catalyst decanted again, and

the liquor was ﬁltered to became clear. After addition of

150 mL of (C₂H₅)₂O under stirring and cooling with an

ice-common salt-mixture dry HCl gas was passed

through the solution until the neutral reaction was

indicated by moistened test paper. It was stirred in the

cooling bath for further 20 min. The precipitate was

collected, washed with (C₂H₅)₂O and dried. Yield 85%

(crude product). The puriﬁcation proceeded by recrys-

tallization from C₂H₅OH. Colourless crystals, mp 167–

168 ^ꢁC (C₂H₅OH) (ref 46: 167–169 ^ꢁC). IR (KBr, cm^ꢀ1):

ꢀ~=1402; 1445; 1503 (NH); 1593 (+NH₂); 1877, 2020;

2168; maxima of a broad band: 2423, 2471, 2560, 2623,

2737, 2808, 2890, 2954; 3217 (NH); 3428 (⁺NH₂). H

NMR (DMSO-d₆, ppm): d=1.67 (s, 4H, –C(4)H₂– and

–C(5)H₂–); 2.99 (s, 4H, –C(3)H₂– and –C(6)H₂–); 3.42

1

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1079

(bs, 2H, NH); 10.26 (very broad and ﬂat singulet, 1H,

HCl). ¹³C NMR (DMSO-d₆, ppm): d=21.30 (C4 and

C5); 44. 66 (C3 and C6). MS (70 eV, 210 ^ꢁC; free base):

m/z (%): 86 (100) [M^+.]; 85 (20); 69 (8), 57 (88), 56 (30).

Anal. [C₄H₁₁ClN₂(122.6)] C, H, N.

d=14.64 (–CH₂–CH₃); 23.84 (C4); 25.47 (C5); 39.64

(–CH₂–CH₃); 47.27 (C3); 48.26 (C6); 180.02 (C¼S).

Anal. [C₇H₁₅N₃S (173.3)] C, H, N.

4.1.4.3. N-Propyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioamide (2d). With n-C₃H₇–NCS. Yield 65%.

Colourless crystals, mp 72–75 ^ꢁC (2-C₃H₇OH). IR (KBr,

4.1.3. 1,2,3,4,5,6-Hexahydropyridazine-1-carbothioamide

.

(2a). 4 mmol (0.488 g) of 1 HCl and 4 mmol (0.392g)

cm^ꢀ1): ꢀ=1486; 1547 (NH); 2869, 2926, 2960 (–CH₃,

~

of KSCN were solved in 4 mL of H₂O and the solvent

was evaporated carefully under gentle boiling. After

cooling the residue was taken up in 1 mL of ice-cold

H₂O. The crystals were collected by aspiration, washed

with small amounts of both H₂O and C₂H₅OH and

recrystallized from C₂H₅OH. Yield 54%. Colourless

crystals, mp 175–176.5 ^ꢁC (C₂H₅OH). IR (KBr, cm^ꢀ1):

ꢀ~=1514 (NH), 1577 (NH₂), 2849, 2930, 2944, 3155

(NH), 3250 (NH_2sym), 3398 (NH_2asym). ¹H NMR

(CDCl₃, ppm): d=1.66 (m, 2H, –C(4)H₂–); 1.80 (m, 2H,

–C(5)H₂–); 2.95 (dt, 2H, –C(3)H₂–, J_-C(3)H-NH=7.3 Hz);

3.35 (t, 1H, NH, J=7.3 Hz); 4.20 (bs, 2H,

–C(6)H2–); 6.64 (bs, 2H, NH₂). ¹³C NMR (CDCl₃,

ppm): d=23.83 (C4); 25.02 (C5); 47.55 (C3); 48.36 (C6);

181.11 (C¼S). MS (70 eV, 30 ^ꢁC): m/z (%): 145 (59)

[M^+.], 86 (12) [M⁺–HSCN], 71 (22), 61 (31) [H₂SCN⁺],

58 (46), 57 (29), 43 (22), 41 (15), 32 (21), 28 (100). Anal.

[C₅H₁₁N₃S (145.2)] C, H, N.

–CH₂–); 3196 (NH); 3299 (NH). ¹H NMR (CDCl₃,

ppm): d=0.95 (t, 3H, –CH₃); 1.62(tq, 2H, –CH ₂–); 1.71

(m, 4H, –C(4)H₂– and –C(5)H₂–); 2.89 (dt, 2H,

–C(3)H₂–); 3.17 (t, 1H, NH); 3.56 (dt, 2H, NH–CH₂–);

4.22 (bs, 2H, –C(6)H₂–); 7.84 (bs, 1H, CS–NH). ¹³C

NMR (CDCl₃, ppm): d=11.49 (CH₃); 22.60 (CH₂); 23.85

(C4); 25.60 (C5); 46.64 (CH₂); 47.34 (C3); 48.39 (C6);

180.54 (C¼S). MS (70 eV, 110 ^ꢁC): m/z (%): 187 (83)

[M^+.], 87 (96), 86 (40) [M^+.–C₃H₇NCS], 71 (51), 61 (20),

59 (26), 58 (86), 57 (43), 43 (60), 42 (17), 41 (70), 39 (19), 30

(100), 28 (39), 27 (30). Anal. [C₈H₁₇N₃S (187.3)] C, H, N.

4.1.4.4. N-Allyl-1,2,3,4,5,6-hexahydropyridazine-1-car-

bothioamide (2e). With CH₂¼CHCH₂–NCS. Yield 74%.

Colourless crystals, mp 105–109 ^ꢁC (2-C₃H₇OH). IR

(KBr, cm^ꢀ1): ꢀ=1483; 1537 (NH); 1638 (C¼C); 2853,

~

2921, 2939 (–CH₂–); 3002( ¼C–H); 3075 (¼CH₂); 3180

(NH); 3305 (NH). ¹H NMR (CDCl₃, ppm): d=1.72(m,

4H, –C(4)H₂– and –C(5)H₂–); 2.91 (dt, 2H, –C(3)H₂–);

3.21 (t, 1H, NH); 4.30 (tt, 2H, allyl; bs: 2H, –C(6)H₂–);

5.17 (m, 2H, allyl); 5.91 (m, 1H, allyl); 7.90 (bs, 1H, CS–

NH). ¹³C NMR (CDCl₃, ppm): d=23.84 (C4); 25.36

(C5); 47.26 (–CH₂–CH¼CH₂); 47.32(C3); 48.45 (C6);

116.15 (–CH₂–CH¼CH₂); 134.38 (–CH₂–CH¼CH₂);

180.22 (C¼S). Anal. [C₈H₁₅N₃S (185.3)] C, H, N.

4.1.4. 1,2,3,4,5,6-Hexahydropyridazine-1-carbothioamides

.

2b–q — general procedure. 5 mmol (0.613 g) of 1 HCl

were suspended in 5 mL of CH₂Cl₂, and after addition

of 5 mmol of (C₂H₅)₃N it was stirred at room temper-

ature. When a clear solution had been formed, a solution

of 5 mmol of the related isothiocyanate solved in 5 mL

of CH₂Cl₂was added dropwise whithin 10 min under

contineously stirring at room temperature, and the stir-

ring was continued for further 20 min. After evapor-

ation of the solvent in vacuum the obtained product was

washed with H₂O, recrystallized from 2-C₃H₇OH and

dried in vacuum. In this manner were obtained.

4.1.4.5. N-Isopropyl-1,2,3,4,5,6-hexahydropyridazien-1-

carbothioamide (2f). With i-C₃H₇-NCS. Yield 50%.

Colourless crystals, mp 94–97 ^ꢁC (2-C₃H₇OH). IR (KBr,

cm^ꢀ1): ꢀ=1476; 1538 (NH); 2853, 2944, 2970 (–CH₃,

~

–CH_2-–, –CH); 3201 (NH); 3285 (NH). ¹H NMR (CDCl₃,

ppm): d=1.22 (d, 6H, 2-CH₃); 1.71 (m, 4H, –C(4)H₂–

and –C(5)H₂–); 2.88 (dt, 2H, –C(3)H₂–); 3.13 (t, 1H,

NH); 4.21 (bs, 2H, –C(6)H₂–); 5.49 (m, 1H, CH); 7.65

(bd, 1H, CS-NH). ¹³C NMR (CDCl₃, ppm): d=22.82

(2-CH₃); 23.90 (C4); 25.69 (C5); 46.32 (CH); 47.33 (C3);

48.32(C6); 179.30 (C ¼S). Anal. [C₈H₁₇N₃S (187.3)] C,

H, N.

4.1.4.1. N-Methyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioamide (2b). With CH₃–NCS. Yield 66%. Col-

ourless crystals, mp 140–142 ^ꢁC (2-C₃H₇OH). IR (KBr,

cm^ꢀ1): ꢀ=1486; 1544 (NH); 2852, 2930 (–CH₃, –CH₂–);

~

3181 (NH); 3287 (NH). ¹H NMR (CDCl₃, ppm):

d=1.69 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.89 (dt, 2H,

–C(3)H₂–); 3.12(d, 3H, –CH ₃); 3.18 (t, 1H, NH); 4.23 (bs,

2H, –C(6)H₂–); 7.83 (bs, 1H, CS–NH). ¹³C NMR (CDCl₃,

ppm): d=23.86 (C4); 25.45 (C5); 31.53 (–CH₃); 47.31

(C3); 48.39 (C6); 181.23 (C¼S). MS (70 eV, 140 ^ꢁC): m/z

(%): 159 (100) [M^+.], 87 (39), 86 (42) [M^+.–CH₃-NCS]

76 (16), 75 (54), 71 (35), 70 (13), 58 (93), 57 (46), 32(13),

30 (68), 28 (40). Anal. [C₆H₁₃N₃S (159.3)] C, H, N.

4.1.4.6. N-Butyl-1,2,3,4,5,6-hexahydropyridazine-1-car-

bothioamide (2g). With n-C₄H₉–NCS. Yield 70%. Col-

ourless crystals, mp 75.5–78 ^ꢁC (2-C₃H₇OH). IR (KBr,

cm^ꢀ1): ꢀ=1492; 1552 (NH); 2858, 2928, 2855 (broad

~

band, –CH₃, –CH₂–); 3181 (NH); 3296 (NH). H NMR

1

(CDCl₃, ppm): d=0.94 (t, 3H, –CH₃); 1.37 (tq, 2H,

–CH₂–); 1.57 (tt, 2H, –CH₂–); 1.71 (m, 4H, –C(4)H₂–

and –C(5)H₂–); 2.89 (dt, 2H, –C(3)H₂–); 3.15 (t, 1H,

NH); 3.59 (dt, 2H, NH–CH₂–); 4.28 (bs, 2H, –C(6)H₂–);

7.80 (bs, 1H, CS–NH). ¹³C NMR (CDCl₃, ppm):

4.1.4.2. N-Ethyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioamide (2c). With C₂H₅-NCS. Yield 64%. Col-

ourless crystals, mp 109–110 ^ꢁC (2-C₃H₇OH). IR (KBr,

cm^ꢀ1): ꢀ=1485; 1546 (NH); 2858, 2938, 2968 (–CH₃,

d=13.92(CH ); 20.25 (CH₂); 23.87 (C4); 25.62 (C5);

~

3

–CH₂–); 3178 (NH); 3288 (NH). ¹H NMR (CDCl₃,

ppm): d=1.21 (t, 3H, –CH₃); 1.69 (m, 4H, –C(4)H₂–

and –C(5)H₂–); 2.90 (dt, 2H, –C(3)H₂–); 3.18 (t, 1H,

NH); 3.62(dq, 2H, NH–C H₂–); 4.21 (bs, 2H, –C(6)H₂–);

7.76 (bs, 1H, CS–NH). ¹³C NMR (CDCl₃, ppm):

31.50 (CH₂); 44.72(CH ₂); 47.36 (C3); 48.40 (C6); 180.75

(C¼S). Anal. [C₉H₁₉N₃S (201.3)] C, H, N.

4.1.4.7. N-(2-Phenylethyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioamide (2h). With C₆H₅–C₂H₄–NCS.

1080

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

Yield 88%. Colourless crystals, mp 115–119 ^ꢁC (2 -

126.37; 128.33; 138.37; 138.93; 179.06 (C¼S). MS (70

eV, 30 ^ꢁC): m/z (%): 235 (19) [M^+.], 149 (15) [C₇H₇-

NCS⁺], 92(20), 87 (31), 86 (11), 58 (38), 57 (17), 32

(46), 30 (22), 28 (100). Anal. [C₁₂H₁₇N₃S (235.4)] C, H,

N.

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1474; 1494, 1542(broad

band, NH, arom. ring); 2856, 2936, 3181 (–CH₂–); 3022,

~

1

3058, 3083 (arom. ¼C–H); 3191 (NH); 3291 (NH). H

NMR (CDCl₃, ppm): d=1.64 (m, 2H, –C(4)H₂–); 1.74

(m, 2H, –C(5)H₂–); 2.81 (dt, 2H, –C(3)H₂–); 2.92 (t, 2H,

–CH₂–C₆H₅); 3.10 (t, 1H, NH); 3.86 (dt, 2H, NH–CH₂–);

4.21 (bs, 2H, –C(6)H₂–); 7.26 (m, 5H, phenyl); 7.85 (bs,

1H, CS–NH). ¹³C NMR (CDCl₃, ppm): d=23.85 (C4);

4.1.4.11. N-(4-Methylphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioamide (2l). With 4-CH₃–C₆H₄–

NCS. Yield 84%. Colourless crystals, mp 89–93 ^ꢁC (2 -

C₃H₇OH, ref 46: 104–107 ^ꢁC). IR (KBr, cm^ꢀ1): ꢀ~=1481,

1527 (broad band, NH, arom. ring); 1583 (arom. ring);

2847, 2915, 2956, 2992 (broad band, –CH₃, –CH₂–);

25.53 (C5); 35.52 (CH₂); 45.92(CH ); 47.25 (C3); 48.37

2

(C6); 126.38; 128.53 (2 C); 128.92 (2 C); 139.30; 180.33

(C¼S). Anal. [C₁₃H₁₉N₃S (249.4)] C, H, N.

1

3022 (arom. ¼C–H); 3209 (NH); 3264 (NH). H NMR

4.1.4.8. N-Cyclohexyl-1,2,3,4,5,6-hexahydropyridazine-

1-carbothioamide (2i). With C₆H₁₁–NCS. Yield 82%.

Colourless crystals, mp 83–88 ^ꢁC (2-C₃H₇OH). IR (KBr,

cm^ꢀ1): ꢀ~=1474; 1533 (NH); 1699; 2850, 2929 (broad

(CDCl₃, ppm): d=1.77 (m, 4H, –C(4)H₂– and –C(5)H₂–);

2.33 (s, 3H, –CH₃); 3.01 (dt, 2H, –C(3)H₂–); 3.40 (t, 1H,

NH); 4.31 (bs, 2H, –C(6)H₂–); 7.15 (d, 2H, p-subst.

phenyl); 7.38 (d, 2H, p-subst. phenyl); 9.62 (s, 1H, CS–

NH). ¹³C NMR (CDCl₃, ppm): d=22.10 (CH₃); 23.78

(C4); 25.34 (C5); 47.56 (C3); 47.98 (C6); 125.09 (2 C);

129.16 (2 C); 135.31; 136.45; 179.41 (C¼S). Anal.

[C₁₂H₁₇N₃S (235.4)] C, H, N.

1

band, –CH₂–, –CH); 3198 (NH); 3282 (NH). H NMR

(CDCl₃, ppm): d=1.24 (m, 4H, cyclohexyl); 1.42 (m,

2H, cyclohexyl); 1.70 (m, 4H, –C(4)H₂– and –C(5)H₂–;

2H, cyclohexyl); 2.04 (m, 2H, cyclohexyl); 2.88 (bs, 2H,

–C(3)H₂–); 3.14 (bs, 1H, NH); 4.19 (m, 2H, –C(6)H₂–;

1H, cyclohexyl); 7.75 (bd, 1H, CS-NH). ¹³C NMR

(CDCl₃, ppm): d=23.83 (cyclohexyl; C4⁰); 24.92 (cyclo-

hexyl; C3⁰, C5⁰); 25.62 (C4); 25.72 (C5); 33.07 (cyclo-

hexyl; C2⁰, C6⁰); 47.26 (C3); 48.32 (C6); 53.00

(cyclohexyl; C1⁰); 179.02(C ¼S). MS (70 eV, 120 ^ꢁC): m/z

(%): 227 (48) [M^+.], 130 (12), 99 (26), 87 (100), 86 (26)

[M^+.ꢀC₆H₁₁NCS], 84 (16), 71 (21), 58 (32), 57 (26), 56

(40), 44 (12), 42 (47), 39 (11). Anal. [C₁₁H₂₁N₃S (227.4)]

C, H, N.

4.1.4.12. N-(2-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioamide (2m). With 2-CH₃O–C₆H₄–

NCS. Yield 81%. Colourless crystals, mp 136–140 ^ꢁC

(2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~=1505, 1539 (broad

band, NH, arom. ring); 1595 (arom. ring); 2835, 2852,

2936 (–CH₃, –CH₂–); 3002, 3064 (arom. ¼C–H); 3155

(NH); 3226 (NH). ¹H NMR (CDCl₃, ppm): d=1.77 (m,

4H, –C(4)H₂– and –C(5)H₂–); 3.03 (m, 2H, –C(3)H₂–);

3.41 (t, 1H, NH);3.85 (s, 3H, –CH₃); 4.33 (bm, 2H,

–C(6)H₂–); 7.00 (md, 3H, o-subst. phenyl); 8.55 (dd, 1H,

o-subst. phenyl); 10.12(bs, 1H, CS–N H). ¹³C NMR

(CDCl₃, ppm): d=23.85 (C4); 25.30 (C5); 47.25 (C3);

47.46 (C6); 55.75 (CH₃); 110.28; 120.16; 123.55; 124.79;

128.52; 150.63; 178.06 (C¼S). Anal. [C₁₂H₁₇N₃OS

(251.4)] C, H, N.

4.1.4.9. N-Phenyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioamide (2j). With C₆H₅–NCS. Yield 83%. Col-

ourless crystals, mp 133–135 ^ꢁC (2-C₃H₇OH, ref 69:

133–134 ^ꢁC). IR (KBr, cm^ꢀ1): ꢀ~=1498; 1533 (broad

band, NH, arom. ring); 1589, 1598 (arom. ring); 2854,

2938 (broad band, –CH₂–); 3182, 3215 (broad band,

arom. ¼C–H, 2ꢄNH). ¹H NMR (CDCl₃, ppm):

d=1.78 (m, 4H, –C(4)H₂– and –C(5)H₂–); 3.02(dt, 2H,

–C(3)H₂–); 3.40 (t, 1H, NH); 4.33 (bs, 2H, –C(6)H₂–);

7.18 (m, 1H, phenyl); 7.35 (m, 2H, phenyl); 7.56 (d, 2H,

phenyl); 9.75 (s, 1H, CS–NH). ¹³C NMR (CDCl₃, ppm):

d=23.78 (C4); 25.16 (C5); 47.53 (C3); 47.77 (C6);

124.57 (2 C); 125.32; 128.49 (2 C);139.06; 178.87 (C¼S).

MS (70 eV, 130 ^ꢁC): m/z (%): 221 (90) [M^+.], 136 (10)

[C₆H₅-NCSH⁺], 135 (12) [M^+.ꢀC₆H₅–NCS], 87 (100),

86 (22), 78 (46), 71 (21), 58 (45), 30 (46), 28 (52). Anal.

[C₁₁H₁₅N₃S (221.3)] C, H, N.

4.1.4.13. N-(4-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioamide (2n). With 4-CH₃O–C₆H₄–

NCS. Yield 34%. Colourless crystals, mp 85–87 ^ꢁC (2 -

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1462, 1525 (broad band,

~

NH, arom. ring); 1586, 1608 (arom. ring); 2834, 2855,

2938 (–CH₃; –CH₂–); 2999, 3047 (arom. ¼C–H); 3238

(NH); 3306 (NH). ¹H NMR (CDCl₃, ppm): d=1.77 (m,

4H, –C(4)H₂– and –C(5)H₂–); 3.01 (m, 2H, –C(3)H₂–);

3.38 (t, 1H, NH);3.81 (s, 3H, –CH₃); 4.32(bs, H2 ,

–C(6)H₂–); 6.89 (d, 2H, p-subst. phenyl); 7.37 (d, 2H, p-

subst. phenyl); 9.53 (bs, 1H, CS–NH). ¹³C NMR

(CDCl₃, ppm): d=23.79 (C4); 25.28 (C5); 47.53 (C3);

48.06 (C6); 55.41 (CH₃); 113.88 (2C); 162.94 (2C);

132.36; 157.41; 177.72 (C¼S). Anal. [C₁₂H₁₇N₃OS

(251.4)] C, H, N.

4.1.4.10. N-(3-Methylphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioamide (2k). With 3-CH₃–C₆H₄–

NCS. Yield 88%. Colourless crystals, mp 93–98 ^ꢁC (2 -

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1484, 1526 (broad band,

~

NH, arom. ring); 1608 (arom. ring); 2853, 2931, 2948,

2993 (broad band, –CH₃, –CH₂–); 3020 (arom. ¼C–H);

3183 (NH); 3236 (NH). ¹H NMR (CDCl₃, ppm):

d=1.78 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.35 (s, 3H,

–CH₃); 3.00 (dt, 2H, –C(3)H₂–); 3.41 (t, 1H, NH);

4.32(bs, 2H, –C(6)H ₂–); 6.99 (d, 1H, m-subst. phenyl);

7.30 (m, 3H, m-subst. phenyl); 9.68 (s, 1H, CS–NH).

¹³C NMR (CDCl₃, ppm): d=22.00 (CH₃); 23.77 (C4);

25.31 (C5); 47.87 (C3); 48.17 (C6); 121.88; 125.37;

4.1.4.14. N-(4-Nitrophenyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioamide (2o). With 4-NO₂–C₆H₄–NCS.

Yield 97%. Yellow crystals, mp 155–158 ^ꢁC (2

-

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1328 (broad multipeak

~

band, ¼C–NO₂), 1515, 1544 (broad band, NH, arom.

ring); 1598 (arom. ring); 2924, 2947 (–CH₂–); 3063

(arom. ¼C–H); 3178 (broad band, 2ꢄNH). H NMR

1

(CDCl₃, ppm): d=1.80 (m, 4H, –C(4)H₂– and –C(5)H₂–

); 3.05 (dt, 2H, –C(3)H₂–); 3.52(t, 1H, NH); 4.35 (bs,

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1081

2H, –C(6)H₂–); 7.95 (dt, 2H, p-subst. phenyl); 8.21 (dt,

2H, p-subst. phenyl); 10.245 (s, 1H, CS–NH). ¹³C

NMR (CDCl₃, ppm): d=23.72 (C4); 25.21 (C5); 47.65

(C3); 47.91 (C6); 121.99 (2 C); 122.48; 124.42 (2 C);

145.11; 178.01 (C¼S). Anal. [C₁₁H₁₄N₄O₂S (266.3)] C,

H, N.

and the reaction occuring at room temperature was

indicated by TLC. Group 1 (Z=5): CH₃NCS (2b),

C₂H₅NCS (2c), H₂C=CH-CH₂NCS (2e), (CH₃)₂CH-

NCS, (2f), C₄H₉NCS (2g); group 2( Z=5): C₃H₇NCS

(2d), C₆H₁₁NCS (2i), C₆H₅NCS (2j), 2-CH₃O–

C₆H₄NCS (2m),4-NO₂-C₆H₄NCS (2o); group 3 (Z=2):

3-CH₃-C₆H₄NCS (2k), naphth-1-yl-NCS (2p). When the

4.1.4.15. N-(Naphth-1-yl)-1,2,3,4,5,6-hexahydropyr-

idazine-1-carbothioamide (2p). With C₁₀H₇–1-NCS.

isothiocyanate was not anymore detectable (iodine azide

72

reaction

on the TLC plate failed), the solvent was

Yield 57%. Beige crystals, mp 119–122 ^ꢁC (2-C₃H₇OH).

evaporated in vacuum, and the residue was solved in

10.00 mL of CH₃CN (ultrasonic bath).

IR (KBr, cm^ꢀ1): ꢀ=1478, 1525 (broad band, NH,

arom. ring); 1594 (arom. ring); 2852, 2935 (–CH₂–);

~

1

3056 (arom. ¼C–H); 3179 (NH); 3227 (NH). H NMR

For the HPLC measurements 1.00 mL of this solution

was taken oﬀ and diluted with CH₃CN up to 10.00 mL.

Injection volume:10 mL.

(CDCl₃, ppm): d=1.78 (tt, 2H, –C(4)H₂–); 1.88 (tt, 2H,

–C(5)H₂–); 3.13 (dt, 2H, –C(3)H₂–); 3.57 (t, 1H,

NH); 4.38 (bs, 2H, –C(6)H₂–); 7.51 (m, 3H, naphth-1-

yl); 7.83 (m, 4H, naphth-1-yl); 9.89 (s, 1H, CS–NH).

Anal. [C₁₅H₁₇N₃S (271.4)] C, H, N.

HPLC. Column: RP-select B (Merck); mobile phase

acetonitrile/0.02M KH ₂PO₄(pH 6.57) 1:1 (v/v). Inves-

tigation conditions: temperature 22 ^ꢁC; ﬂow 0.8

mL min^ꢀ1, time of passage t_odetermined with uracil.

.

4.1.4.16.

N-(Naphth-2-yl)-1,2,3,4,5,6-hexahydropyr-

idazine-1-carbothioamide (2q). With C₁₀H₇–2-NCS.

Yield 78%. Beige crystals, mp 142–148 ^ꢁC (2-C₃H₇OH).

IR (KBr, cm^ꢀ1): ꢀ~=1507, 1525 (broad band, NH,

arom. ring); 1578, 1598, 1631 (arom. ring); 2852, 2917,

2945 (–CH₂–); 3041 (arom. ¼C–H); 3267 (broad band,

NH). ¹H NMR (CDCl₃, ppm): d=1.74 (m, 2H,

–C(4)H₂–); 1.84 (M, 2H, –C(5)H₂–); 3.08 (dt, 2H,

–C(3)H₂–); 3.45 (t, 1H, NH); 4.34 (bs, 2H, –C(6)H₂–);

7.43 (m, 2H, naphth-2-yl); 7.67 (dd, 1H, naphth-2-yl);

7.79 (m, 3H, naphth-2-yl); 8.08 (d, 1H, naphth-2-yl); 9.91

(s, 1H, CS–NH). ¹³C NMR (CDCl₃, ppm): d=23.79

(C4); 25.33 (C5); 47.66 (C3); 47.90 (C6); 121.47; 124.32;

125.37; 126.16; 127.60; 127.69; 128.06; 131.36; 133.51;

136.59; 179.16 (C¼S). MS (70 eV, 280 ^ꢁC): m/z (%): 271

(23) [M^+.], 186 (100) [C₁₀H₇-NCSH⁺], 143 (13), 127

(51) [C₁₀H⁺₇], 87 (44), 86 (15), 58 (24), 30 (27), 28 (12).

Anal. [C₁₅H₁₇N₃S (271.4)] C, H, N.

Detection: Near the absorption maximum utilizing the

uv diode array detector L-7450 (Merck); group 1: 235

nm; group 2: 242 nm; group 3: 249 nm; 2a,h,n: 246 nm;

2l,q: 249 nm. Evaluation: The determination of each k⁰

value (Table 1) for the respective 2 prepared using the

single synthesis mode was done in single runs with 0.01

mM concentrated solutions of these in CH₃CN. The

chromatograms of the combinatorial libraries (groups 1

to 3) were evaluated by means of the k⁰values and by

comparison of the areas under the peaks with those in

the chromatograms of the mixtures of the pure 2 (solved

in CH₃CN, each of the compounds 0.01 mM).

4.1.6. 1,2,3,4,5,6-Hexahydropyridazine-1-carbothioimidic

acid methyl ester hydroiodides 3 — general procedure.

2.5 mmol of the appropriate 2 were solved in 15 mL of

CH₂Cl₂and after addition of 3.75 mmol (0.535 g) of

CH₃I the covered reaction vessel was stored at room

temperature until the starting compound was com-

pletely reacted (24–48 h; reaction control utilizing

TLC). The solvent was carefully evaporated in vacuum;

the formed crystals were suspended in a very small

amount of cold 2-C₃H₇OH, sucked oﬀ, washed with

some 2-C₃H₇OH and dried in vacuum. Non-crystalline

products isolated were frictionized, allowed to stand at

ꢀ8 ^ꢁC until the crystallization was completed and than

they were worked up as described above. In this manner

were obtained:

4.1.4.17. N-Phenyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioamide (2j) and 4-nitrobenzanilide from 1-(4-

nitrobenzoyl)-1,3-diphenylthiourea (6). 1 mmol of 6 and

subsequent 2mmol of (C H₅)₃N were added under stir-

2

.

ring to a suspension of 1 mmol of 1 HCl in 5 mL of

CH₂Cl₂at room temperature. After 10 min the stirring

was ended, and the batch was stored at room temper-

ature for 36 h. After evaporation to dryness with an air

ﬂow a solid residue was yielded, which was suspended in

a small amount of water. The obtained product was

pressed on an earthenplate and after drying it was

recrystallized from C₂H₅OH. The residue yielding by

the latter procedure was washed with a small amount of

C₂H₅OH and dried: 4-nitrobenzanilide,⁷³yield 37%, mp

218—219 ^ꢁC. The crystals formed during the cooling

down of the solution were sucked oﬀ, and recrys-

tallized again form C₂H₅OH. 2j, yield 63%, mp 133–

135 ^ꢁC.

4.1.6.1. 1,2,3,4,5,6-Hexahydropyridazine-1-carbothio-

imidic acid methyl ester hydroiodide (3a). From 2a.

Deviated from the given instruction the non-crystalline

yielded product was dissolved in a small amount of 2-

C₃H₇OH and crystallized by a subsequent careful addi-

tion of (C₂H₅)₂O. Yield 73%. Colourless crystals, mp

102–105 ^ꢁC [2-C₃H₇OH/(C₂H₅)₂O]. IR (KBr, cm^ꢀ1):

~

ꢀ=1478; 1592; 1632 (C¼N); 2851; 2908; 2942; 2957;

1

4.1.5. Combinatorial synthesis of the 2 from 1-hydrochlo-

ride and isothiocyanates. Synthesis of the libraries. A

solution of Z diﬀerent isothiocyanates related to the

respective derivative 2 (each 0.1 mmol in about 1 mL of

CH₂Cl₂) in CH₂Cl₂was added to a mixture of Zꢄ0.1

mmol of 1 hydrochloride and Z 0.1 mmol of (C₂H₅)₃N,

2981; 3110; 3222; 3263; 3302. H NMR (CDCl₃, ppm):

d=1.88 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.94 (s, 3H,

–SCH₃); 3.07 (bs, 2H, –C(3)H₂–); 4.29 (bs, 2H,

–C(6)H₂–); 5.58 (t, 1H, NH); 8.2 9 (bs, 2 H¼, NH^ꢃ₂). ¹³C

NMR (CDCl₃, ppm): ꢁ=17.15 (CH₃), 23.38 (C4); 23.96

:

(C5); 47.38 (C3); 49.36 (C6); 169.78 (C(SCH₃)=NH^ꢃ₂).

1082

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

MS (70 eV, 348 ^ꢁC): m/z (%): 159 (56) [M ], 145 (87)

(C5); 36.32(– CH₂–C₆H₅); 47.42(C3); 47.75 (C6); 51.33

(¼NH^ꢃ–CH₂–); 127.09 (phenyl; C4⁰); 128.84 (phenyl;

C3⁰, C5⁰);⁰); 128.91 (phenyl; C2⁰, C6⁰);⁰); 136.97 (phenyl;

C1⁰); 167.12( C(SCH₃)¼NH^ꢃ–). Anal. [C₁₄H₂₂IN₃S

(391.3)] C, H, N.

+.

base

+.

[M^+.

— CH₂], 142(95) [M

— NH₃], 141 (59),

base

128 (96), 127 (100), 111 (15) [M^+.ꢀꢀCH₃SH], 103 (25),

102(39), 101 (23), 86 (61), 85 (77) [C ₄H₉N⁺₂, M_b⁺_a^._se

—

HN¼C–SCH^.₃], 71 (76), 70 (37), 62(32), 61 (71), 58 (88),

56 (77), 44 (32), 43 (62), 41 (60), 39 (25); 30 (96), 29 (31),

28 (85), 27 (31). Anal. [C₆H₁₄IN₃S (287.2)] C, H, N.

4.1.6.6.

N-Cyclohexyl-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioimidic acid methyl ester hydroiodide

(3i)._ꢁFrom 2i. Yield 67%. Colourless crystals, mp 112–

115 C (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1497; 1530 (NH);

1602(C ¼N); 2858; 2934; 2974; 3070; 3111 (NH), 3140

4.1.6.2. N-Methyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioimidic acid methyl ester hydroiodide (3b). From

2b. Yield 90%. Colourless crystals, mp 98.5–101 ^ꢁC

1

(CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1507; 1620 (C¼N); 2852,

(NH). H NMR (CDCl₃, ppm): d=1.44 (m, 4H, cyclo-

~

2938, 2997 (–CH₃, –CH₂–); 3144 (NH). ¹H NMR

(CDCl₃, ppm): d=1.86 (m, 4H, –C(4)H₂– and –C(5)H₂–);

2.69 (bs, 3H, –SCH₃); 3.11 (bs, 2H, –C(3)H₂–); 3.34 (d,

3H,=NH^ꢃ-CH₃); 4.29 (bs, 2H, –C(6)H₂–); 5.50 (bs, 1H,

NH); 8.85 (bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm):

d=17.67 (–SCH₃); 23.32 (C4); 24.61 (C5); 33.14

(¼NH^ꢃ–CH₃); 47.47 (C3); 51.44 (C6); 167.65

(C(SCH₃)¼NH^ꢃ–). Anal. [C₇H₁₆IN₃S (301.2)] C, H,

N.

hexyl); 1.66 (m, 2H, cyclohexyl); 1.90 (m, 8H, –C(4)H₂–,

–C(5)H₂– and cyclohexyl); 2.75 (s, 3H, –SCH₃); 3.10 (m,

4H, –C(3)H₂– and –CH₂–); 3.94 (m, 1H, ¼NH^ꢃ–CH–);

4.29 (m, 2H, –C(6)H₂–); 5.81 (t, 1H, NH); 8.33 (d, 1H,

¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm): d=18.66 (CH₃);

23.23 (cyclohexyl; C4⁰); 24.43 (C4); 24.61 (cyclohexyl;

C3⁰, C5⁰); 24.76 (C5); 33.56 (cyclohexyl; C2⁰, C6⁰); 47.13

(C3); 51.06 (C6); 56.34 (cyclohexyl; C1⁰); 165.94

(C(SCH₃)¼NH^ꢃ–). Anal. [C₁₂H₂₄IN₃S (369.3)] C, H,

N.

4.1.6.3.

N-Ethyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioimidic acid methyl ester hydroiodide (3c). From

2c. Yield 92%. Colourless crystals, mp 89.5–94 ^ꢁC

(CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1488; 1547 (NH); 1620

(C¼N); 2862; 2938; 2968; 3152 (maximum of a broad

4.1.6.7. N-Phenyl-1,2,3,4,5,6-hexahydropyridazine-1-

carbothioimidic acid methyl ester hydroiodide (3j). From

2j. Yield 76%. Colourless crystals, mp 106.5–110 ^ꢁC

(CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1496; 1578; 1591 (C¼N);

1

band, NH). H NMR (CDCl₃, ppm): d=1.36 (t, 3H,

2940, 3060; 3105 (NH); 3153 (NH). H NMR (CDCl₃,

–CH₃); 1.90 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.72 (bs,

3H, –SCH₃); 3.14 (m, 2H, –C(3)H₂–); 3.77 (dq, 2H,

¼NH^ꢃ–CH₂–); 4.30 (m, 2H, –C(6)H₂–); 5.67 (t, 1H,

NH); 8.69 (bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm):

d=15.65 (–CH₂–CH₃); 18.27 (–S–CH₃); 23.29 (C4);

24.55 (C5); 41.68 (–CH₂–CH₃); 47.43 (C3); 51.25 (C6);

166.95 (C(SCH₃)¼NH^ꢃ–). Anal. [C₈H₁₈IN₃S (315.2)] C,

H, N.

ppm): d=1.92(m, 4H, –C(4)H ₂– and –C(5)H₂–); 2.22 (s,

3H, –SCH₃); 3.19 (m, 2H, –C(3)H₂–); 4.33 (m, 2H,

–C(6)H₂–); 5.78 (bs, 1H, NH); 7.47 (m, 5H, phenyl);

10.18 (bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm):

d=16.80 (–S–CH₃); 23.24 (C4); 24.18 (C5); 47.46 (C3);

51.32 (C6); 125.57 (arom C); 128.42 (arom. C); 129.78

(arom. C); 135.25 (arom. C); 167.78 (C(SCH₃)¼NH^ꢃ–).

Anal. [C₁₂H₁₈IN₃S (363.3)] C, H, N.

4.1.6.4. N-Isopropyl-1,2,3,4,5,6-hexahydropyridazine-

1-carbothioimidic acid methyl ester hydroiodide (3f).

From 2f. Yield 84%. Colourless crystals, mp 101–

4.1.6.8. N-(3-Methylphenyl)-1,2,3,4,5,6-hexahydropyr-

idazine-1-carbothioimidic acid methyl ester hydroiodide

(3k). From 2k. Yield 78%. Colourless crystals, mp

103 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1480; 1536 (NH);

107.5–111 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1498; 1585

~

1604 (C¼N); 2854; 2913; 2940; 3003; 3042; 3070; 3152

(broad band); 2869; 2942; 3009; 3035; 3083; 3090; 3157.

¹H NMR (CDCl₃, ppm): d=1.92(m, 4H, –C(4)H ₂– and

–C(5)H₂–); 2.26 (s, 3H, –SCH₃); 2.39 (s, 3H, –C₆H₄–

CH₃); 3.17 (m, 2H, –C(3)H₂–); 4.36 (m, 2H, –C(6)H₂–);

5.92(bs, 1H, NH); 7.18 (bs, 1H, phenyl); 7.31 (m, 3H,

phenyl); 10.08 (bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃,

ppm): d=16.79 (–S–CH₃); 21.32 (–C₆H₄–CH₃); 23.28

(C4); 24.25 (C5); 47.45 (C3); 51.53 (C6); 122.27 (arom

C); 125.75 (arom C); 129.19 (arom. C); 129.59 (arom

C); 135.18 (arom. C); 140.12(arom. C); 167.67

(C(SCH₃)¼NH^ꢃ–). Anal. [C₁₃H₂₀IN₃S (377.3)] C, H, N.

1

(NH); 3200 (NH). H NMR (CDCl₃, ppm): d=1.39 (d,

6H, 2-CH₃); 1.89 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.76

(s, 3H, –SCH₃); 3.09 (m, 2H, –C(3)H₂–); 4.32(m, 3H,

–C(6)H₂– and ¼NH^ꢃ–CH–); 5.87 (t, 1H, NH); 8.31 (bd,

1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm): d=18.32(–S–

CH₃); 23.21 (C4); 23.51 [–CH(CH₃)₂]; 24.32 (C5);

47.00 (C3); 49.76 (C6); 50.98 [–CH(CH₃)₂]; 165.66

(C(SCH₃)¼NH^ꢃ–). Anal. [C₉H₂₀IN₃S (329.2)] C, H,

N.

4.1.6.5. N-(2-Phenylethyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioimidic acid methyl ester hydroiodide

(3h). From 2h. Yield 71%. Colourless crystals, mp 108–

117 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1504 (NH); 1611

(C¼N); 2854; 2933; 2992; 3090 and 3132 (maxima of a

broad band, NH). ¹H NMR (CDCl₃, ppm): d=1.85 (m,

4H, –C(4)H₂– and –C(5)H₂–); 2.42 (s, 3H, –SCH₃); 3.04

(m, 4H, –C(3)H₂– and –CH₂–); 3.98 (dt, 2H, ¼NH^ꢃ–

CH₂–); 4.23 (m, 2H, –C(6)H₂–); 5.74 (t, 1H, NH); 7.28

(m, 5H, phenyl); 8.91 (bs, 1H, ¼NH^ꢃ–). ¹³C NMR

(CDCl₃, ppm): d=17.80 (–SCH₃); 23.24 (C4); 24.60

4.1.6.9. N-(4-Methylphenyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioimidic acid methl ester hydroiodide (3l).

From 2l. Yield 74%. Colourless crystals, mp 101–108 ^ꢁC

(CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1510 (NH); 1679 (C¼N);

~

2860; 2935; 3019; 3081; 3070; 3181 (NH). ¹H NMR

(CDCl₃, ppm): d=1.92(m, 4H, –C(4)H

– and

2

–C(5)H₂–); 2.24 (s, 3H, –SCH₃); 2.38 (s, 3H, –C₆H₄–

CH₃); 3.18 (m, 2H, –C(3)H₂–); 4.33 (m, 2H, –C(6)H₂–);

5.92 (bs, 1H, NH); 7.23 (d, 2H, phenyl); 7.41 (d, 2H,

phenyl); 10.02(bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃,

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1083

ppm): d=16.77 (–S–CH₃); 21.16 (–C₆H₄–CH₃); 23.26

(C4); 24.23 (C5); 47.48 (C3); 51.38 (C6); 125.36 (2 arom.

C); 130.34 (2arom. C); 132.59 (arom. C); 138.64 (arom.

C); 167.63 (C(SCH₃)¼NH^ꢃ–). Anal. [C₁₃H₂₀IN₃S

(377.3)] C, H, N.

(CDCl₃, ppm): d=16.81 (–S–CH₃); 23.28 (C4); 24.22

(C5); 47.52 (C3); 51.56 (C6); 123.08; 123.98; 127.23; 127.56;

127.87; 128.06; 129.90; 132.34; 132.63; 133.13; 168.21

(C(SCH₃)¼NH^ꢃ–). Anal. [C₁₆H₂₀IN₃S (413.3)] C, H, N.

4.1.7.

amide hydroiodides 4a–v.

1,2,3,4,5,6-Hexahydropyridazine-1-carboximid-

4.1.6.10. N-(2-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioimidic acid methyl ester hydro-

iodide (3m). From 2m. Yield 75%. Colourless crystals,

mp 118.5–123 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1499;

Method A — from diﬀerent 3 and gaseous NH₃

1

1582(C ¼N); 1600; 2923; 2952; 2975; 3083; 3219. H

Into a solution of 1 mmol of the related 1,2,3,4,5,6-

hexahydropyridazine-1-carbothioimidic acid methyl

ester hydroiodide 3 dissolved in 4 mL of CH₂Cl₂NH₃

was bubbled for 5 min and then the covered batch

(pressure compensation!) was allowed to stand at room

NMR (CDCl₃, ppm): d=1.98 (m, 4H, –C(4)H₂– and

–C(5)H₂–); 2.31 (s, 3H, –SCH₃); 3.20 (m, 2H, –C(3)H₂–

); 3.91 (s, 3H, –C₆H₄–OCH₃); 4.43 (m, 2H, –C(6)H₂–);

6.23 (bs, 1H, NH); 7.02 (m, 2H, phenyl); 7.34 (m, 1H,

phenyl); 7.56 (d, 1H, phenyl); 9.64 (bs, 1H, ¼NH^ꢃ–).

¹³C NMR (CDCl₃, ppm): d=16.92(–S– CH₃); 22.97

(C4); 24.45 (C5); 47.45 (C3); 51.13 (C6); 56.00 (–O–

CH₃); 111.73 (arom C); 121.16 (arom. C); 126.79 (arom.

C); 130.11 (2arom C); 153.33 (arom. C); 168.40

(C(SCH₃)¼NH^ꢃ–). Anal. [C₁₃H₂₀IN₃OS (393.3)] C, H,

N.

temperarature for 2h. Thereafter (C H₅)₂O was care-

2

fully added until the solution became turbidly, and after

allowing to stand for a longer time crystals precipitated

which were aspirated oﬀ, washed with (C₂H₅)₂O/

2-C₃H₇OH (3:1; V:V) and dried in vacuum. In this

manner were obtained:

4.1.7.1.

1,2,3,4,5,6-Hexahydropyridazine-1-carbox-

4.1.6.11. N-(4-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carbothioimidic acid methyl ester hydro-

iodide 3n. From 2n. Yield 69%. Colourless crystals, mp

102–110 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ~=1513; 1597

(C¼N, NH); 2940; 3055; 3125 (NH). ¹H NMR (CDCl₃,

ppm): d=1.91 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.24

(s, 3H, –SCH₃); 3.17 (m, 2H, –C(3)H₂–); 3.38 (s, 3H,

–C₆H₄–OCH₃); 4.33 (m, 2H, –C(6)H₂–); 5.86 (bs, 1H,

NH); 6.94 (m, 2H, phenyl); 7.45 (m, 2H, phenyl); 9.97

(bs, 1H, ¼NH^ꢃ–). ¹³C NMR (CDCl₃, ppm): d=16.56

(–S–CH₃); 23.34 (C4); 24.15 (C5); 47.17 (C3); 51.54

(C6); 55.59 (–O–CH₃); 114.87 (4 arom C); 127.02 (2

imidamide hydroiodide (4a). From 3a. Yield 60%. Col-

ourless crystals, mp 140–143 ^ꢁC (CH₂Cl₂/(C₂H₅)₂O). IR

(KBr, cm^ꢀ1): ꢀ~=1598; 1640 (C¼N); 2848; 2921; 2939;

3158; 3217; 3316; 3428. ¹H NMR (DMSO-d₆, ppm):

d=1.56 (m, 4H, –C(4)H₂– and –C(5)H₂–); 2.79 (m, 2H,

–C(3)H₂–); 3.52(m, 2H, –C(6)H ₂–); 5.17 (t, 1H, NH);

7.19 (s, 4H, –C(NH₂)¼NH^ꢃ₂). ¹³C NMR (DMSO-d₆,

ppm): d=23.20 (C4); 23.38 (C5); 44.87 (C3); 46.39 (C6);

156.30 (C(NH₂)¼NH₂^ꢃ). MS (70 eV, 348 ^ꢁC): m/z (%):

128 (100) [M^+._base, HI^+.], 127 (55), 86 (30) [C₄H₁₀N₂^+.

,

M^+.

(10), 30 (27), 28 (37). Anal. [C₅H₁₃IN₄(256.1)] C, H, N.

— HN¼C¼NH], 85 (13), 57 (38), 43 (27), 41

base

arom.

C);

159.49

[C₁₃H₂₀IN₃OS (393.3)] C, H, N.

(C(SCH₃)¼NH^ꢃ–).

Anal.

4.1.7.2. N²-Methyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4b). From 3b. Yield 58%.

Colourless, markedly hygroscopic crystals, mp 41–47 ^ꢁC

(CH₂Cl₂/(C₂H₅)₂O). IR (KBr, cm^ꢀ1): ꢀ~=1570; 1631

(C¼N); 2856; 2940; 2939; 3212 and 3298 (maxima of a

4.1.6.12. N-(Naphth-1-yl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioimidic acid methyl ester hydroiodide

(3p). From 2p. Yield 75%. Yellowish crystals, mp 89.5–

ꢁ

95 C (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1478; 1509; 1589

broad band). H NMR (DMSO-d₆, ppm): d=1.60 (m,

1

~

(very intensive; C¼N and aromatic moiety); 2821; 2861;

4H, –C(4)H₂– and –C(5)H₂–); 2.76 (d, 3H, CH₃); 2.79

(m, 2H, –C(3)H₂–); 3.53 (m, 2H, –C(6)H₂–); 5.13 (t, 1H,

NH); 7.49 (s, 2H, NH₂); 7.70 (q, 1H, CH₃NH¹¼). ¹³C

NMR (DMSO-d₆, ppm): d=23.19 (C4); 23.51 (C5); 28.20

(CH₃); 45.21 (C3); 46.22 (C6); 155.88 (C(NH₂)¼NH^ꢃ–).

1

2926; 2942; 2976; 3015; 3075; 3179. H NMR (CDCl₃,

ppm), spectrum of the free base: d=1.94 (m, 2H,

–C(4)H₂–); 2.03 (m, 2H, –C(5)H₂–); 2.11 (s, 3H, –CH₃);

3.25 (t, 2H, –C(3)H₂–); N(2)H is not to detect; 4.47

(t, 2H, –C(6)H₂–); 7.49–7.73 (m, 4H, naphth-1-yl); 7.91

(m, 1H, naphth-1-yl); 7.93 (m, 1H, naphth-1-yl); 8.32(d,

1H, naphth-1-yl). Anal. [C₁₆H₂₀IN₃S (413.3)] C, H, N.

MS (70 eV, 120 ^ꢁC): m/z (%): 142(35) [M _base], 128 (66),

+.

127 (31), 86 (90) [C₄H₁₀N⁺₂^., M

— HN¼C¼N–CH₃],

+.

base

85 (19), 57 (100), 56 (15), 43 (16), 41 (13), 30 (34), 28

(26). Anal. [C₆H₁₅IN₄(270.1)] C, H, N.

4.1.6.13. N-(Naphth-2-yl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carbothioimidic acid methyl ester hydroiodide

(3q). From 2q. Yield 60%. Yellowish crystals, mp 93.5–

4.1.7.3. N²-(2-Phenylethyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carboximidamide hydroiodide (4h). From 3h.

Yield 87%. Colourless crystals, mp 109.5–112 ^ꢁC

(CH₂Cl₂/(C₂H₅)₂O). IR (KBr, cm^ꢀ1): ꢀ~=1497; 1566;

1642(C ¼N); 2855; 2884; 2916; 2932; 2946; 2960; 3026;

98 ^ꢁC (CH₂Cl₂). IR (KBr, cm^ꢀ1): ꢀ=1476; 1496; 1511;

~

1589 (very intensive; C¼N and aromatic moiety); 2857;

2942 (maximum of a multipeak band); 3032; 3090, 3110.

¹H NMR (CDCl₃, ppm), spectrum of the free base:

d=1.87 (m, 2H, –C(4)H₂–); 1.98 (m, 2H, –C(5)H₂–); 2.23

(s, 3H, –CH₃); 3.19 (t, 2H, –C(3)H₂–); N(2)H is not to

detect; 4.39 (t, 2H, –C(6)H₂–); 7.54–7.59 (m, 2H, naphth-

1-yl); 7.64 (dd, 1H, naphth-1-yl); 7.84–7.92(m, 3H,

naphth-1-yl); 8.00 (d, 1H, naphth-1-yl). ¹³C NMR

1

3167, 3297; 3331. H NMR (DMSO-d₆, ppm): d=1.58

(bs, 4H, –C(4)H₂– and –C(5)H₂–); 2.76 (m, 2H, –C(3)H₂–);

2.80 (t, 2H, ¼NH^ꢃ–CH₂–CH₂–C₆H₅); 3.41 (t, 2H,

¼NH^ꢃ–CH₂–CH₂–C₆H₅); 3.52(m, 2H, –C(6)H ₂–); 5.16

(t, 1H, NH); 7.27 (m, 5H, arom. H); 7.56 (s, 2H, NH₂);

7.68 (t, 1H, ¼NH–CH₂–CH₂–C₆H₅). ¹³C NMR

1084

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

+.

(DMSO-d₆, ppm): d=23.23 (C4); 23.48 (C5); 34.49

(–CH₂–CH₂–C₆H₅); 42.31 (–CH₂–CH₂–C₆H₅); 45.30

(C3); 46.21 (C6); 126.32; 128.27; 128.83; 138.32; 155.02

(–C(NH₂)¼NH^ꢃ–CH₂–CH₂–C₆H₅). MS (70 eV, 348 ^ꢁC):

m/z (%):232 (30) [M^+._base], 204 (26), 197 (12), 195 (12),

193 (22), 189 (13), 183 (22), 182 (12), 181 (22), 172 (15),

157 (15), 155 (15), 146 (12), 141 (31), 139 (17), 128 (39),

127 (21), 120 (41), 119 (100) [C₆H₅–CH₂–CH¼NH^+.],

105 (40), 104 (34), 103 (23), 102 (49), 101 (13), 92 (54),

(%): 156 (30) [M ], 128 (92), 127 (44), 86 (100)

base

[C₄H₁₀N⁺₂^., M

— HN¼C¼N–C₂H₅], 85 (19), 72

+.

base

(12), 57 (54), 56 (12), 44 (23), 43 (47), 41 (13), 32 (20), 30

(33), 29 (14), 28 (100). Anal. [C₇H₁₇IN₄(284.1)] C, H, N.

4.1.7.6. N¹-Isopropyl-1,2,3,4,5,6-hexahydropyridazine-

1-carboximidamide hydroiodide (4f). From 3f. Yield

62%. Colourless crystals, mp 134–136.5 ^ꢁC (2

-

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1559; 1619; 1638

~

91 (27), 86 (82) [C₄H₁₀N⁺₂^.,M^+.

— HN¼C¼N–

(C¼N); 2856; 2928; 2940; 2974; 3182, 3193; 3297. H

1

base

CH₂–CH₂–C₆H₅], 85 (19), 79 (12), 78 (11), 77 (24), 76

(28), 75 (24), 66 (16), 58 (11), 57 (25), 56 (11), 52 (10), 51

(17), 50 (15), 43 (12), 41 (15), 39 (12), 30 (23), 28 (25).

Anal. [C₁₃H₂₁IN₄(360.2)] C, H, N.

NMR (DMSO-d₆,

ppm):

d=1.16

(d,

6H,

–CH(CH₃)₂); 1.60 (bs, 4H, –C(4)H₂– and –C(5)H₂–);

2.79 (m, 2H, –C(3)H₂–); 3.54 (m, 2H, –C(6)H₂–); 3.77

(m, 1H, NH–CH(CH₃)₂); 5.14 (t, 1H, NH); 7.28 (d, 1H,

NH–CH(CH₃)₂); 7.56 (s, 2H, ¼NH^ꢃ₂). ¹³C NMR

(DMSO-d₆, ppm): d=22.14 (–CH(CH₃)₂); 23.22 (C4);

23.51 (C5); 43.61 (–CH(CH₃)₂); 45.23 (C3); 46.25 (C6);

154.32(– C(NH^ꢃ₂)–NH–CH(CH₃)₂). MS (70 eV, 348 ^ꢁC):

4.1.7.4. N²-(4-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4k). From

3k. Yield 65%. Colourless crystals, mp 138–141 ^ꢁC

(CH₂Cl₂/(C₂H₅)₂O). IR (KBr, cm^ꢀ1): ꢀ=1512; 1594;

m/z (%): 170 (40) [M ], 86 (100) [C₄H₁₀N⁺₂^., M

—

+.

base

~

base

1634 (C¼N); 2845; 2938; 3105; 3196; 3280; 3426; 3449.

¹H NMR (DMSO-d₆, ppm): d=1.65 (bs, 4H, –C(4)H₂–

and –C(5)H₂–); 2.88 (bs, 2H, –C(3)H₂–); 3.67 (bs, 2H,

–C(6)H₂–); 3.78 (s, 3H,-OCH₃); 5.33 (t, 1H, NH); 7.01

(d, 2H, arom. H); 7.17 (d, 2H, arom. H); 7.40 (bs, 2H,

NH₂); 9.36 (bs, 1H, ¼NH^ꢃ–C₆H₄–OCH₃). ¹³C NMR

(DMSO-d₆, ppm): d=23.24 (C4); 23.41 (C5); 45.60

(C3); 46.41 (C6); 55.30 (–OCH₃); 114.61 (2C), 127.72;

127.85; 155.06; 158.05 (–C(NH₂)¼NH^ꢃ–). MS (70 eV,

HN¼C¼N–CH(CH₃)₂], 85 (32), 57 (60), 56 (15), 44

(29), 43 (25), 41 (19), 32 (11), 30 (25), 28 (58). Anal.

[C₈H₁₉IN₄(298.2)] C, H, N.

4.1.7.7. N²-(2-Phenylethyl)-1,2,3,4,5,6-hexahydropyrid-

azine-1-carboximidamide hydroiodide (4h). From 3h.

Yield 30%. For analytical data see 4.1.7.3.

4.1.7.8. N²-Phenyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4i). From 3i. Yield 30%.

Yellowish crystals, mp 153.5–156 ^ꢁC (2-C₃H₇OH). IR

348 ^ꢁC): m/z (%): 234 (37) [M^+._base], 148 (25) [M^+.

base

— C₄H₁₀N₂], 147 (12), 133 (15), 128 (30), 127 (18), 123

(12), 92 (10), 86 (100) [C₄H₁₀N⁺₂^., M^+.

(KBr, cm^ꢀ1): ꢀ=1497; 1520; 1604; 1634; 1642 (C¼N);

~

—

HN¼C¼N–C₆H₄–OCH₃], 85 (14), 77 (12), 57 (26), 30

base

2940; 3036; 3083, 3148; 3179; 3250; 3370. ¹H NMR

(DMSO-d₆, ppm): d=1.64 (m, 2H, –C(4)H₂–); 1.71 (m,

2H, –C(5)H₂–); 2.91 (m, 2H, –C(3)H₂–); 3.66 (m, 2H,

–C(6)H₂–); 5.34 (t, 1H, NH); 7.24 (m, 2H, arom. H);

7.33 (m, 1H, arom. H); 7.46 (m, 2H, arom. H); 7.63 (bs,

2H, NH₂); 9.51 (s, 1H, ¼NH^ꢃ–C₆H₅). ¹³C NMR

(DMSO-d₆, ppm): d=23.27 (C4); 23.42 (C5); 45.74

(C3); 46.49 (C6); 125.29 (broad signal, phenyl, C3⁰, C5⁰);

126.49 (phenyl, C4⁰); 129.43 (phenyl, C2⁰, C6⁰); 135.64

(phenyl, C1⁰); 154.64 (–C(NH₂)¼NH^ꢃ–C₆H₅). MS (70

(16), 28 (11). Anal. [C₁₂H₁₉IN₄O (362.2)] C, H, N.

Method B — from diﬀerent 3 and aqueous solution of NH₃

To a solution of 1.5 mmol of the related 1,2,3,4,5,6-

hexahydropyridazine-1-carbothioimidic acid methyl

ester hydroiodide 3 in 5 mL of CH₃OH were added 2

mL of a 25 percentage aqueous solution of NH₃. Then

the covered batch (pressure compensation!) was allowed

to stand at room temperarature for 2d. After the sol-

vent was evaporated carefully, the dry product yielded

was stored in a refrigerator to allow it to crystallize; the

crystals were suspended in a small amount of 2-

C₃H₇OH, then they were separated, recrystallized from

2-C₃H₇OH, sucked oﬀ, washed and dried in vacuum. In

this manner were obtained:

ꢁ

+.

eV, 345 C): m/z (%): 204 (56) [M ], 128 (64), 127

base

(29), 119 (38) [H₂N–C¼N–C₆H⁺₅], 93 (14), 86 (100)

[C₄H₁₀N⁺₂^., M

(11), 77 (54), 58 (48), 56 (13), 51 (18), 43 (10), 41 (13), 30

(25), 28 (17). Anal. [C₁₁H₁₇IN₄(332.2)] C, H, N.

— HN¼C¼N–C₆H₅], 85 (22), 71

+.

base

4.1.7.9. N²-(2-Methoxyphenyl)-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4j). From 3j.

Yield 35%. Yellowish crystals, mp 135–137 ^ꢁC (2 -

4.1.7.5. N²-Ethyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4c). From 3c. Yield 50%.

Colourless crystals, mp 123–126 ^ꢁC (2-C₃H₇OH). IR

(KBr, cm^ꢀ1): ꢀ~=1496; 1615 and 1641 (NH, C¼N,

maxima of an intensive band); 2922; 2946; 2979; 3175,

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1502; 1578; 1599; 1640

~

1

(C¼N); 2916; 2884; 2946; 3172; 3305; 3437; 3538. H

NMR (DMSO-d₆, ppm): d=1.65 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.87 (m, 2H, –C(3)H₂–); 3.65 (m, 2H,

–C(6)H₂–); 3.81 (s, 3H, –OCH₃); 5.37 (t, 1H, NH); 7.01

(m, 1H, arom. H); 7.22 (m, 2H, arom. H); 7.35 (s, 2H,

NH₂); 7.37 (m, 1H, arom. H); 9.11 (s, 1H, ¼NH^ꢃ–

C₆H₄–OCH₃). ¹³C NMR (+ DEPT 135) (DMSO-d₆,

ppm): d=23.28 (C4); 23.43 (C5); 45.63 (C3); 46.40 (C6);

55.61 (–O–CH₃); 112.50; 120.68; 123.15; 128.70; 129.16;

154.49; 155.01 (C(NH₂)¼NH^ꢃ-). MS (70 eV, 40 ^ꢁC): m/z

1

3204 and 3293 (maxima of a broad band). H NMR

(DMSO-d₆, ppm): d=1.08 (t, 3H, –CH₂–CH₃), 1.60 (m,

4H, –C(4)H₂– and –C(5)H₂–); 2.79 (m, 2H, –(3)H₂–);

3.20 (dq, 2H, –NH–CH₂–CH₃); 3.54 (m, 2H, –C(6)H₂–);

5.15 (t, 1H, NH); 7.52(s, H2 , NH ₂); 7.68 (t, 1H,

¼NH^ꢃ–CH₂–CH₃). ¹³C NMR (DMSO-d₆, ppm):

d=14.43 (–CH₂–CH₃); 23.27 (C4); 23.55 (C5); 35.95

(–CH₂–CH₃); 45.25 (C3); 46.27 (C6); 155.05

(ꢀC(NH₂)¼NH^ꢃ–CH₂–CH₃). MS (70 eV, 100 ^ꢁC): m/z

+.

base

(%): 234 (50) [M ], 148 (14), 142 (11), 134 (27), 128

(65), 127 (35), 123 (13), 120 (13), 105 (10), 92 (15), 87

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1085

(14), 86 (100) [C₄H₁₀N⁺₂^., M

— HN¼C¼N–C₆H₄–

Yield 80%. Colourless crystals, mp 100–105 ^ꢁC

+.

base

OCH₃], 85 (30), 77 (15), 65 (11), 58 (68), 57 (18), 56 (19),

43 (14), 41 (14), 30 (34), 28 (19). Anal. [C₁₂H₁₉IN₄O

(362.2)] C, H, N.

[(C₂H₅)₂O]. IR (KBr, cm^ꢀ1): ꢀ=1560; 1619; 1639

~

1

(C¼N); 2855; 2943; 3162, 3218; 3270; 3298; 3357. H

NMR (DMSO-d₆, ppm): d=0.88 (t, 3H, –CH₃); 1.27

(tq, 2H, –CH₂–); 1.44 (tt, 2H, –CH₂–); 1.60 (bs, 4H,

–C(4)H₂– and –C(5)H₂–); 2.77 (m, 2H, –C(3)H₂–); 3.16

(dt, 2H, ¼NH^ꢃ–CH₂–); 3.54 (m, 2H, –C(6)H₂–); 5.15 (t,

Method C — from 3a and diﬀerent amines

To a solution of 1 mmol of 3a in 4 mL of CH₂Cl₂was

added 1 mmol of the related amine, and the covered

batch (pressure compensation!) was allowed to stand at

room temperature for 5 d. Then the residue obtained by

evaporation of the solvent was dried at 110 ^ꢁC for 1 h.

After cooling down to room temperature the non-crys-

talline product was covered with a small amount of

(C₂H₅)₂O, and it was allowed to stand until the crystal-

lization was completed. The formed crystals were sus-

pended in (C₂H₅)₂O, collected by aspiration, washed

with (C₂H₅)₂O/2-C₃H₇OH (3:1; V:V), and dried in

vacuum. In this manner were obtained:

1H, NH); 7.52(s, 2H, –NH ); 7.64 (t, 2H, ¼NH^ꢃ–CH₂–).

2

¹³C NMR (DMSO-d₆, ppm): d=13.55 (–CH₃); 19.15

(–CH₂–); 23.23 (C4); 23.53 (C5); 30.63 (–CH₂–); 40.75

(¼NH^ꢃ–CH₂–); 45.26 (C3); 46.25 (C6); 155.13

(–C(NH₂)¼NH^ꢃ–CH₂–CH₂–CH₂–CH₃). MS (70 eV,

348 ^ꢁC): m/z (%): 184 (30) [M^+._base], 159 (17), 145 (61),

+.

base

142(94) [M

— H₂C¼CH–CH₃], 141 (16), 139 (14),

128 (19), 127 (31), 98 (17), 97 (10), 86 (100) [C₄H₁₀N₂^+.

,

+.

base

M

— HN¼C¼N–C₄H₉], 85 (20), 84 (10), 71 (11), 57

(50), 56 (19), 55 (12), 43 (35), 42 (12), 41 (24), 30 (34), 29

(16), 28 (33), 27 (13). Anal. [C₉H₂₁IN₄(312.2)] C, H, N.

Method D—from diﬀerent 3 with aqueous solution of

CH₃NH₂

4.1.7.10. N²-Propyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4d). With n-C₃H₇–NH₂.

Yield 65%. Colourless crystals, mp 83–90 ^ꢁC

To a solution of 2mmol of the related 1,2,3,4,5,6-hexa-

hydropyridazine-1-carbothioimidic acid methyl ester

hydroiodide 3 in 5 mL of CH₃OH were added 2mL of a

40percentage aqueous solution of CH₃NH₂and the

covered batch (pressure compensation!) was allowed to

stand at room temperature for 1 d. The solvent was

evaporated with slightly warming to dryness and the

obtained residue was stored in the refrigerator until

the crystallization started. The formed crystals were sus-

pended in a small amount of 2-C₃H₇OH, then seper-

ated, recrystallized from 2-C₃H₇OH, washed, and dried

in vacuum. In this manner were obtained:

[(C₂H₅)₂O]. IR (KBr, cm^ꢀ1): ꢀ=1490; 1558; 1617

~

1

(C¼N); 2869; 2916; 2955; 3159, 3190; 3310; 3325. H

NMR (DMSO-d₆, ppm): d=0.85 (t, 3H, –CH₂–CH₂–

CH₃), 1.50 (tq, 2H, –CH₂–CH₂–CH₃); 1.60 (bs, 4H,

–C(4)H₂– and –C(5)H₂–); 2.79 (m, 2H, –C(3)H₂–); 3.13

(dt, 2H, –NH–CH₂–CH₂–CH₃); 3.54 (m, 2H,

–C(6)H₂–); 5.16 (t, 1H, NH); 7.52(s, 2H, NH ₂); 7.67 (t,

1H, ¼NH^ꢃ–CH₂–CH₂–CH₃). ¹³C NMR (DMSO-d₆,

ppm): d=10.79 (–CH₂–CH₂–CH₃); 21.86 (–CH₂–CH₂–

CH₃); 23.19 (C4); 23.52 (C5); 42.50 (–CH₂–CH₂–CH₃);

45.24 (C3); 46.22 (C6); 155.12 (–C(NH₂)¼NH^ꢃ–CH₂–

CH₂–CH₃). MS (70 eV, 347 ^ꢁC): m/z (%): 170 (26)

[M ], 128 (28), 127 (14), 86 (100) [C₄H₁₀N⁺₂^., M

—

4.1.7.13. N¹,N²-Dimethyl-1,2,3,4,5,6-hexahydropyrid-

azine-1-carboximidamide hydroiodide (4l). From 3b.

Yield 45%. Colourless crystals, mp 118–119 ^ꢁC (2 -

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~=1495; 1615; 1625

+.

base

+.

base

HN¼C¼N–C₃H₇], 85 (25), 58 (12), 57 (49), 56 (13), 55

(12), 43 (50), 41 (21), 30 (22), 28 (29), 27 (11). Anal.

[C₈H₁₉IN₄(298.2)] C, H, N.

1

(C¼N); 2850; 2939; 2974; 3048; 3083; 3169; 3252. H

4.1.7.11. N²-Allyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4e). With CH₂¼CH–

CH₂–NH₂. Yield 60%. Colourless crystals, mp 88–96 ^ꢁC

NMR (DMSO-d₆, ppm): d=1.57 (m, 2H, –C(4)H₂–);

1.67 (m, 2H, –C(5)H₂–); 2.83 [bd, 3H (CH₃) and 2 H

(–C(3)H₂–)]; 3.45 (m, 2H, –C(6)H₂–); 5.08 (t, 1H, NH);

7.48 (s, 2H, ¼NH^ꢃ–CH₃). ¹³C NMR (DMSO-d₆, ppm):

d=23.48 and 23.57 (C4 and C5); 30.31 (–CH₃); 46.51

(C3); 47.11 (C6); 158.92( C(NH–CH₃)=NH^ꢃ–). MS (70

eV, 170 ^ꢁC): m/z (%): 202 (45), 201 (65), 177 (24), 162

[(C₂H₅)₂O]. IR (KBr, cm^ꢀ1): ꢀ=1565; 1614; 1641

~

1

(C¼N); 2854; 2919; 2945; 3153, 3211; 3298. H NMR

(DMSO-d₆, ppm): d=1.62(bs, 4H, –C(4)H ₂– and

–C(5)H₂–); 2.80 (m, 2H, –C(3)H₂–); 3.56 (m, 2H,

–C(6)H₂–); 3.84 (m, 2H, ¼NH^ꢃ–CH₂–CH¼CH₂); 5.12

(bs, 1H, NH); 5.20 (m, 2H, –CH₂–CH¼CH₂); 5.82(m,

1H, –CH₂–CH¼CH₂); 7.52(s, 2H, NH ₂); 7.92(t, 2H,

¼NH^ꢃ–CH₂–CH¼CH₂). ¹³C NMR (DMSO-d₆, ppm):

d=23.23 (C4); 23.45 (C5); 42.72 (–CH₂–CH¼CH₂);

45.33 (C3); 46.29 (C6); 115.53 (–CH₂–CH¼CH₂);

+.

(13), 156 (11) [M ], 128 (24), 127 (11), 124 (20), 122

— CH₃–

base

(17), 105 (97), 86 (41) [C₄H₁₀N⁺₂^., M

+.

base

N¼C¼N–CH₃], 78 (19), 77 (59), 72(10), 57 (27), 51

(28), 47 (13), 43 (17), 41 (10), 32 (26), 30 (12), 28 (100).

Anal. [C₇H₁₇IN₄(284.1)] C, H, N.

133.62(–CH ¼CH¼CH₂); 155.17 (–C(NH₂)¼NH–

4.1.7.14. N¹-Ethyl-N²-methyl-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4m). From

3c. Yield 55%. Colourless crystals, mp 98–100.5 ^ꢁC

2

CH₂–CH¼CH₂). MS (70 eV, 348 ^ꢁC): m/z (%): 168 (26)

+.

base

[M ], 128 (80), 127 (40), 99 (20), 86 (100) [C₄H₁₀N₂^+.

,

+.

base

~

M

83 (18), 71 (16), 57 (50), 56 (31), 55 (12), 43 (21), 41 (48),

— HN¼C¼N–CH₂–CH¼CH₂], 85 (27), 84 (14),

(2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1605 (C¼N);

2869; 2939; 3076, 3179 and 3248 (maxima of a broad

band). ¹H NMR (DMSO-d₆, ppm): d=1.14 (t, 3H,

–CH₂–CH₃); 1.58 (m, 2H, –C(4)H₂–); 1.68 (m,

2H, –C(5)H₂–); 2.84 [bd, 3H (¼NH^ꢃ–CH₃) and

2H (–C(3)H₂–)]; 3.24 (dq, 2H, –NH–CH₂–CH₃); 3.47

(m, 2H, –C(6)H₂–); 5.12(t, 1H, NH); 7.34 (t, 1H,

39 (18), 30 (26), 28 (24). Anal. [C₈H₁₇IN₄(296.2)] C, H,

N.

4.1.7.12. N²-Butyl-1,2,3,4,5,6-hexahydropyridazine-1-

carboximidamide hydroiodide (4g). With n-C₄H₉–NH₂.

1086

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

–NH–CH₂–CH₃); 7.53 (q, 1H, CH₃NH^ꢃ¼). ¹³C NMR

(DMSO-d₆, ppm): d=14.92(–CH ₂–CH₃); 23.56 (C4

and C5); 30.50 (–CH₂–CH₃and ¼NH^ꢃ–CH₃); 46.57

(C3); 47.35 (C6); 158.33 (C(NH–CH₃)¼NH^ꢃ–). MS (70

7.08 (m, 2H, arom. H); 7.18 (m, 1H, arom. H); 7.41 (m,

2H, arom. H); 8.39 (q, 1H, –NH–CH₃); 9.50 (s, 1H,

¼NH^ꢃ–C₆H₅). ¹³C NMR (DMSO-d₆, ppm): d=23.39

(C4); 23.41 (C5); 30.24 (–NH–CH₃); 46.55 (C3); 47.94

(C6); 120.50 (phenyl, C3⁰, C5⁰); 124.22 (phenyl, C4⁰);

129.43 (phenyl, C2⁰, C6⁰); 138.08 (phenyl, C1⁰); 155.29

(–C(NHCH₃)¼NH–C₆H₅). MS (70 eV, 350 ^ꢁC): m/z

eV, 300 ^ꢁC): m/z (%): 170 (9) [M ], 128 (44), 127 (19),

+.

base

86 (100) [C₄H₁₀N⁺₂^., M

— CH₃–N¼C¼N–C₂H₅], 85

+.

base

(34), 78 (19), 57 (64), 30 (20), 28 (39). Anal. [C₈H₁₉IN₄

(298.2)] C, H, N.

— C₄H₉N^.₂], 132

+.

base

+.

(%): 218 (41) [M ], 133 (69) [M

base

(14), 128 (57), 127 (31), 118 (31), 92 (10), 87 (10), 86

(100) [C₄H₁₀N⁺₂^., M

(17), 77 (39), 58 (39), 51 (12), 41 (11), 30 (18), 28 (15).

Anal. [C₁₂H₁₉IN₄(346.2)] C, H, N.

+.

base

4.1.7.15. N²-Methyl-N¹-propyl-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4n). From

3d, which resulted in form of a non-crystalline semisolid

mass from the reaction of 2d and CH₃I according the

general procedure for the preparation of the 1,2,3,4,5,6-

hexahydropyridazine-1-carbothioimidic acid methyl

esters 3. Yield 85%. Colourless crystals, mp 94.5–

96.5 ^ꢁC (2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~?=1498, 1609

— CH₃–N¼C¼N–C₆H₅], 85

4.1.7.18. N¹-Methyl-N²-(4-methylphenyl)-1,2,3,4,5,6-

hexahydropyridazine-1-carboximidamide hydroiodide (4r).

From 3l. Yield 30%. Colourless crystals, mp 177–

178 ^ꢁC (2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~=1512; 1562;

1

(C¼N), 2871, 2920, 2942, 2965, 3096. 3157, 3276. H

1624; 1637 (C¼N); 2856; 2936; 3152; 3209; 3305. H

NMR (DMSO-d₆, ppm): d=0.87 (t, 3H, –CH₂–CH₂–

CH₃), 1.55 [m, 2H (–CH₂–CH₂–CH₃) and 2H (–C(4)H₂–)];

1.67 (m, 2H, –C(5)H₂–); 2.83 [bd, 3H (¼NH^ꢃ–CH₃) and

2H (–C(3)H₂–)]; 3.15 (dt, 2H, –NH–CH₂–CH_2--CH₃);

3.45 (m, 2H, –C(6)H₂–); 5.12(t, 1H, NH); 7.33 (t,

1H, –NH–CH₂–CH₂–CH₃); 7.52(q, 1H, ¼NH^ꢃ–CH₃).

¹³C NMR (+ DEPT 135) (DMSO-d₆, ppm): d=10.92

(–CH₂–CH₂–CH₃); 22.35 (–CH₂–CH₂–CH₃); 23.57 (C4,

C5); 30.54 (¼NH^ꢃ–CH₃); 45.18 (–NH–CH₂–CH₂–

CH₃); 46.57 (C3); 47.35 (C6); 158.48 (C(NH-

NMR (DMSO-d₆, ppm): d=1.59 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.28 (s, 3H, –C₆H₄–CH₃); 2.66 (d, 3H, –

NH–CH₃); 2.88 (bs, 2H, –C(3)H₂–); 3.35 (bs, 2H,

–C(6)H₂–); 5.32(t, 1H, NH); 6.97 (d, 2H, arom. H);

8.29 (q, 1H, –NH–CH₃); 9.38 (s, 1H, ¼NH^ꢃ–C₆H₄–

CH₃). ¹³C NMR (+ DEPT 135) (DMSO-d₆, ppm):

d=20.29 (Ar–CH₃); 23.40 (C4); 23.47 (C5); 30.19

(–NH–CH₃); 46.57 (C3); 47.88 (C6); 120.88 (2 C); 129.83

(2C); 133.62; 135.40; 155.42(– C(NHCH₃)¼NH^ꢃ–). MS

(70 eV, 270 ^ꢁC): m/z (%): 232 (17) [M^+._base], 147 (26)

CH₃)¼NH^ꢃ–). MS (70 eV, 350 ^ꢁC): m/z (%): 184 (9)

[M

— C₄H₉N^.₂], 132 (11), 128 (26), 127 (12), 91 (14),

+.

base

[M ], 128 (42), 127 (23), 99 (12), 86 (100) [C₄H₁₀N₂^+.

M

,

86 (100) [C₄H₁₀N⁺₂^., M

— CH₃–N¼C¼N–C₆H₄–

+.

base

+.

base

+.

base

— CH₃–N¼C¼N–C₃H₇], 85 (15), 70 (16), 57 (77),

CH₃], 85 (10), 84 (10), 57 (22), 30 (13), 28 (18). Anal.

[C₁₃H₂₁IN₄(360.2)] C, H, N.

56 (10), 43 (12), 41(16), 30 (25), 28 (19), 27 (10). Anal.

[C₉H₂₁IN₄(312.2)] C, H, N.

4.1.7.19. N²-(2-Methoxyphenyl)-N¹-methyl-1,2,3,4,5,6-

hexahydropyridazine-1-carboximidamide hydroiodide (4s).

From 3m. Yield 35%. Colourless crystals, mp 130–

4.1.7.16.

N²-Methyl-N¹-(2-phenylethyl)-1,2,3,4,5,6-

hexahydropyridazine-1-carboximidamide hydroiodide (4p).

From 3h. Yield 65%. Colourless crystals, mp 108–

134 ^ꢁC (2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1504; 1577,

~

109 ^ꢁC (2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ=1589; 1631

1601; 1629; 2834; 2850; 2918; 2937; 3040; 3126; 3213. ¹H

NMR (DMSO-d₆, ppm): d=1.56 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.59 (d, 3H, –NH–CH₃); 2.83 (bs, 2H, –

C(3)H₂–); 3.38 (bs, 2H, –C(6)H₂–); 3.84 (s, 3H, –OCH₃);

5.31 (t, 1H, NH); 6.97 (d, 2H, arom. H); 7.07 (d, 2H,

arom. H); 8.17 (q, 1H, –NH–CH₃); 9.31 (s, 1H, ¼NH^ꢃ–

C₆H₄–OCH₃). ¹³C NMR (DMSO-d₆, ppm): d=23.17

(C4); 23.42 (C5); 29.79 (–NH–CH₃); 46.57 (C3); 47.59

(C6); 55.80 (–OCH₃); 112.13; 120.92; 124.87; 126.25

(tert. arom. C); 127.40; 152.20 (tert. arom. C); 156.46

(–C(NHCH₃)¼NH^ꢃ–). MS (70 eV, 350 ^ꢁC): m/z (%):

~

1

(C¼N); 2852; 2918; 2935; 2950; 3001; 3168; 3260. H

NMR (CDCl₃, ppm): d=1.71 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.87 (bs, 2H, –C(3)H₂–); 2.93 (d, 3H,

(¼NH^ꢃ–CH₃); 3.02(t, H2, –NH–CH ₂–CH₂–C₆H₅);

3.69 (dt, 2H, –NH–CH₂–CH₂–C₆H₅); 3.79 (bs, 2H,

–C(6)H₂–); 4.47 (m, 1H, NH); 6.77 (t, 1H, –NH–CH₂–

CH₂–C₆H₅); 6.99 (q, 1H, ¼NH^ꢃ–CH₃); 7.29 (m, 5H,

arom. H). ¹³C NMR (+ DEPT 135) (DMSO-d₆, ppm):

d=23.48 (C4); 23.51 (C5); 30.28 (¼NH^ꢃ–CH₃); 34.96

(–CH₂–CH₂–C₆H₅); 44.81 (–CH₂–CH₂–C₆H₅); 46.54

(C3); 47.26 (C6); 126.34; 128.30 (2 C); 128.72 (2 C);

138.25; 158.31 (–C(NH^ꢃ–CH₃)¼NH–). MS (70 eV,

+.

248 (30) [M ], 203 (49) [M

— CH₃–NH–CH₃], 202

base

+.

(31), 163 (27) [M

— C₄H₉N^.₂], 162(14), 148 (31), 147

(62), 146 (12), 142 (26), 134 (17), 133 (25), 128 (87), 127

base

370 ^ꢁC): m/z (%): 246 (8) [M ], 128 (35), 127 (19), 105

+.

base

(31), 91 (13), 86 (100) [C₄H₁₀N⁺₂^., M

— CH₃-

(58), 112 (15), 120 (22), 119 (24), 105 (17), 100 (15), 92

(18), 86 (100) [C₄H₁₀N⁺₂^., M

+.

base

+.

base

N¼C¼N–C₂H₄–C₆H₅], 85 (11), 70 (57), 57 (26), 30 (36),

— CH₃–N¼C¼N–

28 (19). Anal. [C₁₄H₂₃IN₄(374.3)] C, H, N.

C₆H₄–OCH₃], 85 (16), 84 (10), 78 (16), 77 (14), 71 (20),

65 (12), 58 (28), 56 (12), 51 (12), 44 (11), 42 (17), 41

(14), 30 (27), 28 (22). Anal. [C₁₃H₂₁IN₄O (376.2)] C, H,

N.

4.1.7.17. N¹-Methyl-N²-phenyl-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4q). From 3j.

Yield 55%. Yellowish crystals, mp 172–177 ^ꢁC (2 -

C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~=1496; 1562; 1592; 1631

(C¼N); 2854; 2938; 2979; 3038; 3155, 3196. ¹H

NMR (DMSO-d₆, ppm): d=1.60 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.69 (d, 3H, –CH₃); 2.92 (m, 2H,

–C(3)H₂–); 3.40 (m, 2H, –C(6)H₂–); 5.37 (t, 1H, NH);

4.1.7.20. N²-(4-Methoxyphenyl)-N¹-methyl-1,2,3,4,5,6-

hexahydropyridazine-1-carboximidamide hydroiodide (4t).

From_ꢁ3n. Yield 55%. Colourless crystals, mp 129–

131.5 C (2-C₃H₇OH). IR (KBr, cm^ꢀ1): ꢀ~=1514; 1574;

1612; 1643; 2862; 2941; 3012; 3044; 3167; 3262; 3321. ¹H

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

1087

NMR (DMSO-d₆, ppm): d=1.60 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.64 (d, 3H, –NH–CH₃); 2.89 (bs, 2H,

(DMSO-d₆, ppm): d=23.60 (2 C, C4 and C5); 24.85

[2C, N(CH –CH₂–CH₂–CH₂)]; 46.58 (C3); 47.40 (C6);

2

–C(3)H₂–); 3.42(bs, H2, –C(6)H

₂–); 3.78 (s, 3H,

49.53 [2C, N( CH₂–CH₂–CH₂–CH₂)]; 156.76 (C¼NH^ꢃ₂).

MS (70 eV, 348 ^ꢁC): m/z (%): 182(44) [M

_base], 128

+.

–OCH₃); 5.30 (t, 1H, NH); 7.14 (m, 4H, arom. H);

8.16 (q, 1H, –NH–CH₃); 9.05 (s, 1H, ¼NH^ꢃ–C₆H₄–

OCH₃). ¹³C NMR (+ DEPT 135; CDCl₃, ppm):

d=23.90 (C4); 24.01 (C5); 31.34 (–NH–CH₃); 47.57

(C3); 49.34 (C6); 55.54 (–O–CH₃); 114.81 (2C); 124.95

(2C); 129.07; 156.33; 157.92(– C(NHCH₃)¼NH^ꢃ–). MS

(81), 127 (35), 112 (15), 97 (30), 86 (100) [C₄H₁₀N₂^+.

,

M^+.— C₄H₈N–CN], 85 (27), 84 (10), 71 (75)

[C₄H₈N^+., M^+.— C₄H₉N₂–CN], 57 (50), 56 (14), 55

(55), 43 (27), 42 (19), 41 (25), 30 (27), 28 (50), 27 (11).

Anal. [C₉H₁₉IN₄(310.2)] C, H, N.

ꢁ

+.

(70 eV, 350 C): m/z (%): 248 (21) [M ], 163 (28)

[M

base

— C₄H₉N^.₂], 162(15), 147 (15), 146 (15), 133 (11),

4.1.7.23. N²-Methyl-N¹,N¹-tetramethylene-1,2,3,4,5,6-

+.

base

128 (21), 127 (12), 86 (100) [C₄H₁₀N⁺₂^., M

— CH₃–

hexahydropyridazine-1-carboximidamide

hydroiodide

(4v). From 3b and pyrrolidine. Reaction time 5 days.

+.

base

N¼C¼N–C₆H₄–OCH₃], 57 (20), 28 (42). Anal.

[C₁₃H₂₁IN₄O (376.2)] C, H, N.

Yield 50%. Colourless crystals, mp 165–171 ^ꢁC. IR

(KBr, cm^ꢀ1): ꢀ=1595 (C¼N); 2852; 2929; 2970;

~

Method E — from diﬀerent 3 and diﬀerent amines

3056; 3151; 3226. ¹H NMR (DMSO-d₆, ppm):

d=1.56 (m, 2H, –C(4)H₂–); 1.72(m, H2 , –C(5)H ₂–);

1.86 [m, 4H, N(CH₂–CH₂–CH₂–CH₂)]; 2.83 [bd, 3H

(¼NH^ꢃ–CH₃) and 2H (–C(3)H₂–)]; 3.45 [m, 2H

(–C(6)H₂–) and 4H (N(CH₂–CH₂–CH₂–CH₂)]; 5.10

(t, 1H, NH); 7.38 (q, 1H, ¼NH^ꢃ–CH₃). ¹³C NMR

(DMSO-d₆, ppm): d=23.56 (C4); 23.74 (C5); 24.74 [2 C,

N(CH₂–CH₂–CH₂–CH₂)]; 31.13 (¼NH^ꢃ–CH₃); 46.39

(C3); 48.39 (C6); 48.96 [2C, N( CH₂–CH₂–CH₂–CH₂)];

157.42( C¼NH^ꢃ–CH₃). MS (70 eV, 348 ^ꢁC): m/z (%):

To a solution of 1 mmol of the related 1,2,3,4,5,6-hexa-

hydropyridazine-1-carbothioimidic acid methyl ester

hydroiodide 3 in 3 mL of CH₂Cl₂was added 1 mmol of

the appropriate amine and the covered batch (pressure

compensation!) was allowed to stand at room temper-

ature. After the reaction was completed, the solvent was

removed with slightly warming to dryness and the resi-

due obtained was stored in the refrigerator until the

crystallization started. The formed crystals were pressed

oﬀ on an earthenplate, then they were suspended in a

small amount of (C₂H₅)₂O/2-C₃H₇OH (9:1; V/V),

sucked oﬀ, washed with some (C₂H₅)₂O/2-C₃H₇OH

(9:1; V/V), and dried in vacuum. In this manner were

obtained:

+.

196 (10) [M ], 128 (39), 127 (28), 111 (11), 110 (100)

base

[C₆H₁₀N⁺₂^.

,

M^+.—C₄H₁₀N₂], 71 (13), 70 (38)

[C₄H₈N⁺], 57 (11), 55 (13), 42(12), 41 (13), 30 (13), 28

(15). Anal. [C₁₀H₂₁IN₄(324.2)] C, H, N.

4.1.8. 1-(4-Nitrobenzoyl)-1,3-diphenylthiourea (6). 0.01

mol of 1,3-diphenylthiourea was solved in 30 mL of

CH₂Cl₂with slightly warming, and it was added a warm

solution of 0.01 mol of 4-nitrobenzoylchloride in 30 mL

of CH₂Cl₂. The batch was allowed to stand at room

temperature for 12h, and an intensiv evolution of HCl

occured. The solution was ﬁltered and than at room

temperature concentrated in vacuum. The obtained

non-crystalline yellow coloured residue was grinded

with n-hexane until the crystallization started and then

the crystals were digested for four times with each 10

mL of boiling n-hexane. The product was sucked oﬀ,

washed with n-hexane and dried on an earthenplate.

Yield 81%. Bright yellow crystals. To prevent a quick

decomposition the substance has to store proper

enclosed and at a cool place, mp 123–129 ^ꢁC (n-hexane;

decomposition and recrystallisation are to observe

between 130 and 135 ^ꢁC, and beginning at about 195 ^ꢁC

sublimation takes place; the sublimate representing

4-nitrobenzanilide melts at 218–219 ^ꢁC; reference:⁷³

4.1.7.21. N²-Methyl-N¹-butyl-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4o). From 3b

and n-C₄H₉NH₂. Reaction time 1 day. Yield 85%. Col-

ourless crystals, mp 47.5–53 ^ꢁC. IR (KBr, cm^ꢀ1):

~

ꢀ=1612, 1626 (maxima of an intensive band); 2857;

2873; 2933; 2952; 3107; 3170; 3285. H NMR (DMSO-

1

d₆, ppm): d=0.89 (t, 3H, –CH₃); 1.29 (tq, 2H, –CH₂–);

1.53 (tt, 2H, –CH₂–); 1.67 (bs, 4H, –C(4)H₂– and

–C(5)H₂–); 2.83 [bd, 3H+2H, ¼NH^ꢃ–CH₃(J=4.2

Hz)+ –C(3)H₂–]; 3.18 (dt, 2H, –NH–CH₂–); 3.46 (m,

2H, –C(6)H₂–); 5.09 (t, 1H, NH); 7.32(t, 1H, NH–);

7.53 (q, 1H, ¼NH^ꢃ–CH₃, J=4.2Hz). ¹³C NMR

(DEPT 135. DMSO-d₆, ppm): d=13.48 (–CH₃); 19.12

(–CH₂–); 23.55 and 23.53 (C4, C5); 30.47 (¼NH^ꢃ–

CH₃); 31.04 (–CH₂–); 43.20 (–NH–CH₂–); 46.58 (C3);

47.35 (C6); 158.45 (C¼NH^ꢃ–). MS (70 eV, 200 ^ꢁC): m/z

+.

(%): 198 (8) [M ], 128 (26), 127 (12), 86 (100)

base

[C₄H₁₀N⁺₂^., M

— CH₃–N¼C¼N–C₄H₉], 85 (12), 57

+.

base

(53), 41 (12), 30 (14), 29 (10), 28 (16). Anal. [C₁₀H₂₃IN₄

(326.2)] C, H, N.

216–217 ^ꢁC). IR (KBr, cm^ꢀ1): ꢀ=1490; 1523; 1592 (NH);

~

1669 (CO); maxima of a broad band: 3028, 3119, 3226,

1

3321. H NMR (CDCl₃, ppm): d=7.24–8.32 (m, 14H,

4.1.7.22. N¹,N¹-Tetramethylene-1,2,3,4,5,6-hexahydro-

pyridazine-1-carboximidamide hydroiodide (4u). From 3a

and pyrrolidine. Reaction time 3 days. Yield 40%. Col-

arom. H); 12.50 (bs, 1H, NH). ¹³C NMR (DMSO-d₆,

ppm): d=123.18; 124.58; 125.16; 127.07; 128.21; 128.98;

129.03; 129.52; 130.59; 138.10; 140.49; 141.93; 147.86;

171.51 (C¼S); 182.32 (C¼O). Anal. [C₂₀H₁₅N₃O₃S

(377.4)] C, H, N.

ourless crystals, mp 90–94 ^ꢁC. IR (KBr, cm^ꢀ1): ꢀ=1550;

~

1603; 1653 (C¼N); 2852; 2870; 2906; 2947; 3168; 33308;

3364. ¹H NMR (DMSO-d₆, ppm): d=1.55 (m,

2H, –C(4)H₂–); 1.69 (m, 2H, –C(5)H₂–); 1.85 [m, 4H,

N(CH₂–CH₂–CH₂–CH₂)]; 2.82 (m, 2H, –C(3)H₂–); 3.43

[m, 2H (–C(6)H₂–) and 4H (N(CH₂–CH₂–CH₂–CH₂)];

4.98 (t, 1H, NH); 7.57 (s, 2H, ¼NH^ꢃ₂). ¹³C NMR

4.2. Biology

4.2.1. Cell line. The rat insulinoma cell line RIN-5AH,⁷⁴

an adherent b-cell line, was grown in plastic culture

1088

O. Morgenstern et al. / Bioorg. Med. Chem. 12 (2004) 1071–1089

ﬂasks (Greiner, Frickenhausen, Germany). Cells were

maintained in RPMI-1640 medium (Biochrom, Berlin,

Germany) containing 2mmol/l L-glutamine, 100 U/mL

penicillin, 100 mg/mL streptomycin and 5% heat-inacti-

vated fetal calf serum at 37 ^ꢁC in a 5% CO₂atmosphere.

For the experiment cells were trypsinized, washed twice

with Hanks medium and counted. Viability of cells was

determined by trypan blue exclusion test.

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50

Acknowledgements

The authors wish to thank the Deutsche For-

schungsgemeinschaft for the ﬁnancial support of this

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Synthesis, structural investigations and biological evaluation of novel hexahydropyridazine-1-carboximidamides, -carbothioamides and -carbothioimidic acid esters as inducible nitric oxide synthase inhibitors

DOI: 10.1016/j.bmc.2003.12.007

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