electrospray mass spectrometric studies of noncovalent complexes of buspirone hydrochloride and...

RAPID COMMUNICATIONS IN MASS SPECTROMETRY

Rapid Commun. Mass Spectrom. 2003; 17: 2139–2146

Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/rcm.1166

Electrospray mass spectrometric studies of noncovalent

complexes of buspirone hydrochloride and other

serotonin 5-HT1A receptor ligands containing

arylpiperazine moieties

Piotr Kowalski1, Piotr Suder2, Teresa Kowalska1, Jerzy Silberring2, Beata Duszynska3

and Andrzej J. Bojarski3*1Institute of Organic Chemistry and Technology, Cracow University of Technology, 24 Warszawska Street, 31-155 Cracow, Poland2Neurobiochemistry Group, Faculty of Chemistry and Regional Laboratory, Jagiellonian University, 3 Ingardena Street, 30-060 Cracow, Polandand Center for Polymer Chemistry, Polish Academy of Sciences, Zabrze, Poland3Department of Medicinal Chemistry, Institute of Pharmacology Polish Academy of Sciences, 12 Smetna Street, 31-343 Cracow, Poland

Received 9 May 2003; Revised 23 July 2003; Accepted 23 July 2003

Noncovalent complexes consisting of two protonated amines and a chloride anion were observed

under electrospray ionization mass spectrometry (ESI-MS) conditions. The observed phenomenon

was investigated for the hydrochlorides of buspirone, a well-known anxiolytic drug, and 23 other

arylpiperazine derivatives that had been developed as serotonin 5-HT1A receptor ligands. Due to

the major role of ionic interactions in a vacuum, it was proposed that the detected complexes

were formed by NHþ��Cl��NHþ bridges. It was found that complexation depended on structural

features of the analyzed compounds. For derivatives with a shorter linker (three methylene groups)

containing a terminal cyclic amide fragment, complex ions were not observed. It was postulated

that, in the latter case, steric hindrance due to a terminal group could disturb ionic bridge forma-

tion. Since both the observed complexation and ligand-binding processes are driven by noncova-

lent forces, and a qualitative relationship between them was found (compounds with a 4-carbon

chain always display higher affinity for 5-HT1A receptors than do their 3-carbon analogues), such

ESI-MS studies may yield valuable information on ligand–receptor interactions. Copyright # 2003

John Wiley & Sons, Ltd.

Electrospray ionization mass spectrometry (ESI-MS) has

recently been applied in our laboratory to characterize

some newly synthesized ligands of serotonin 5-HT1A recep-

tors. When these amines in the form of free bases were ana-

lyzed, their positive ESI-MS spectra contained only the

respective [MþH]þ ion peaks. In the case of their hydro-

chloride salts, however, complex ions of more than double

the molecular mass were often also found. The same phenom-

enon was observed for the hydrochloride of buspirone (1), a

well-known anxiolytic drug.1 To the best of our knowledge,

detection of such complex ions of amine hydrochlorides

under ESI conditions has not yet been reported in the litera-

ture. Here we present the results of experiments undertaken

to determine the composition of these complex ions, features

involved in their formation, as well as some hypotheses about

their structure.

Buspirone and the other related compounds under study

belong to the largest and most thoroughly investigated

arylpiperazine class of serotonin 5-HT1A receptor ligands.2–4

Their general chemical structure consists of an alkyl chain (3–

4 methylene units) attached to the N4 atom of the piperazine

moiety, and a terminal amide or imide fragment.4 Besides

their affinity for serotonin receptors, such compounds are

also often ligands of different subtypes of dopamine and

adrenergic receptors.5,6 They constitute a source of potential

therapeutic agents for the treatment of anxiety, depression,

psychoses, and other central nervous system dysfunctions.

The 5-HT1A receptor belongs to a superfamily of G-protein

coupled receptors (GPCR) whose precise three-dimensional

(3-D) structure is still not known. The available data indicate

that the binding site of both the endogenous neurotransmitter

(serotonin, 5-HT) and of exogenous ligands is located in the

central pocket formed by seven transmembrane a-helical

fragments.7 It is generally accepted that the main interaction

involves formation of an ionic bond between the conserved

aspartic acid on helix three and the protonated amine group

of the ligand.8

Despite a wide range of successful applications of the ESI-

MS technique, it cannot be used directly to study ligand–

GPCR complexes because the structure and function of these

proteins depend on the membrane environment. With regard

to the compounds active at serotonin receptors, ESI-MS was

Copyright # 2003 John Wiley & Sons, Ltd.

*Correspondence to: A. J. Bojarski, Department of MedicinalChemistry, Institute of Pharmacology Polish Academy ofSciences, 12 Smetna Street, 31-343 Cracow, Poland.E-mail: [email protected]

recently used in structural metabolic studies of 1-(3-[5-{1,2,4-

triazol-4-yl}-1H-indol-3-yl]propyl)-4-(2-{3-fluoropheny-

l}ethyl)piperazine, a 5-HT1D agonist.9 It was also applied in

combination with liquid chromatography (LC) for the

determination of antimigraine drugs in human serum,10 to

in vitro studies of metabolism of the 5-HT3 receptor

antagonists tropisetron, ondansetron and dolasetron,11 and

to characterize serotonin derivatives in frog-skin secretions.12

EXPERIMENTAL

Synthesis of new compounds 8b and 12bCompounds 8b and 12bwere obtained using the same proce-

dure as described previously.13 Base 8b was obtained in 58%

yield, m.p. 79–828C (acetone/H2O 5:1); 1H-NMR (90 MHz,

CDCl3): d 1.58–2.02 (m, 4H, CH2CH2CH2CH2), d 2.35–2.56

(m, 6H, CH2N(CH2)2 and CH2N(CH2)2), d 3.78–3.98 (m, 6H,

(CH2)2NAr and CH2NC O), d 6.53 (t, 1H, 5HPyrim, J¼ 4.7

Hz), d 6.98–7.35 (m, 4Harom), d 8.34 (d, 2H, 4HPyrim and

6HPyrim, J¼4.7 Hz); Hydrochloride: m.p. 215–2188C (etha-

nol/acetone 1:10). Anal. Calcd. for C19H23N5O2�HCl�H2O

(407.90): C, 55.95; H, 6.42; N, 17.16. Found: C, 55.96; H, 6.20; N,

17.09.

Base 12b was obtained as an oil in 61% yield; 1H-NMR (90

MHz, CDCl3): d 1.61–1.89 (m, 4H, CH2CH2CH2CH2), d 2.42–

2.58 (m, 6H, CH2N(CH2)2 and CH2N(CH2)2), d 3.76–3.98 (m,

6H, (CH2)2NAr and CH2NC–O), d 4.60 (s, 2HBenzox, CH2), d6.49 (t, 1H, 5HPyrim, J¼ 4.7 Hz), d 6.92–7.10 (m, 4HArom), d 8.31

(d, 2H, 4HPyrim and 6HPyrim, J¼ 4.7 Hz); Hydrochloride: m.p.

210–2138C (ethanol/acetone 2:1). Anal. Calcd. for

C20H25N5O2�HCl (403.91): C, 59.47; H, 6.49; N, 17.34. Found:

C, 59.20; H, 6.36; N, 17.48.

Mass spectrometry measurementsMass spectra were obtained in the positive-ion mode using a

Finnigan MAT 95S double-focusing sector instrument (Finni-

gan MAT, Bremen, Germany) with reversed geometry [B, E]

(B¼magnetic field, E¼ electric field), equipped with an ESI

source. The instrument was tuned and operated as described

previously.14,15 Resolution was adjusted to 1200 (10% valley).

Each sample was dissolved in methanol at a concentration of

50–150 ng/mL and delivered by direct infusion with a syr-

inge pump (Harvard type 22; South Natick, MA, USA) at a

flow rate of 25 mL/min. During the preliminary experiments,

several solvents were tested to mimic the ’real-life’ system,

e.g., methanol, acetonitrile, 30% methanol/water, 30% aceto-

nitrile/water, and water. As no differences in the results and

complex formation were observed, pure methanol was

selected. The ESI interface was operated as follows: tempera-

ture of the heated capillary was set at 2008C, and the spray

voltage was 2.5 kV. Magnetic scanning over the m/z range

1200–50 was used at a scan rate of 5 s/decade. For the MS/

MS experiments (B/E linked scanning measuring product

ions) collisions were performed using helium in the collision

chamber located in the first field-free region (FFFR) of the

mass spectrometer. The intensity of the precursor ions was

decreased to approximately 30% of their initial abundance.

RESULTS AND DISCUSSION

The majority of the compounds studied (2–12) were pre-

viously identified as serotonin 5-HT1A and/or 5-HT2A recep-

tor ligands with high potency in vitro.13,16,17 Systematic

modifications of their structures involved an aryl fragment,

the length of an alkenyl linker (3 or 4 units, sets a and b,

respectively) and a terminal part (benzoxazinone, benzoxa-

zolinone or benzoxazolindione moieties).

As mentioned in the Introduction, in some ESI mass spectra

of the hydrochlorides of 1–12, complex ions with masses

higher than the [MþH]þ ion of the respective base were

detected. A full-scan ESI mass spectrum of the hydrochloride

salt of 1, representative of that group, is shown in Fig. 1(A). It

shows a strong signal at m/z 386, corresponding to the

[MþH]þ ion of the free base 1, and a peak at m/z 807 with

approximately 22% relative abundance. The expanded

spectrum of the region around m/z 807 (Fig. 1(B)) shows a

typical isotopic pattern for ions containing one chlorine atom.

The MS/MS experiment utilizing ions at m/z 807 as

precursors (Fig. 1(C)) yielded product ions at m/z 386. Thus

both these species atm/z 386 appear to correspond to the same

molecular form of base 1. In view of the fact that the mass

difference between ions at m/z 807 and 386 is 421 Da, and that

a chlorine atom was present in the molecular formula of m/z

of 807, it may be concluded that the peak at m/z 807 can be

identified as a [2MþHClþH]þ complex. Taking into

account that the amine hydrochlorides were dissociated in

the bulk solution before electrospraying, so that both cations

(protonated base) and chloride anions were present, it seems

that the formula [2(MþH)þþCl�]þ may more adequately

reflect the structure of the complex. Similar chloride-bound

cluster ions ([X–Cl�–Y]þ, where X, Y¼Naþ, Kþ, Csþ and

Rbþ) are known from studies devoted to estimation of

heterolytic bond dissociation energies of inorganic salts.18

Nominal masses of all the [2(MþH)þþCl�]þ complexes

found are presented in Table 1.

After determining the complex composition, experiments

were designed to explore its nature and to identify features

involved in its formation. First, it was found that the complex

composition did not depend on the amount of hydrochloride

in the analyzed sample, since the ESI spectra of mono- and

dihydrochloride salts of compound 10bwere identical. It was

also observed that the solvent applied for an ESI analysis

(methanol, acetonitrile, acetonitrile/water methanol/water

or water) did not influence the pattern of the spectra. Next,

neither the solute concentration, nor the presence of water

molecules in the analyzed sample (3a, 4b, 7b and 8b),

correlated with the presence of [2(MþH)þþCl�]þ signals.

On the other hand, taking account of the structural features of

the analyzed compounds, it was easy to notice that complexes

were detected only for compounds with a 4-carbon linker (set

b). No complexes were detected for all five compounds with

the shorter chain (2a, 3a, 5a, 7a and 9a). Thus it seems that the

complex formation crucially depends on the alkenyl spacer

length.

Additional experiments were performed with hydrochlor-

ide mixtures of buspirone and compounds 7b or 7a, contain-

ing four- and three-membered spacers, respectively (Fig. 2).

As expected, in the first case, a signal at m/z 803, correspond-

ing to the complex of formula [(1þH)þþ(7bþH)þþCl�]þ,

was observed. Interestingly, in the other spectrum, the

corresponding heterogeneous [(1þH)þþ(7aþH)þþCl�]þ

complex was also detected, but its intensity was low, despite

Copyright # 2003 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2003; 17: 2139–2146

2140 P. Kowalski et al.

Figure 1. ESI mass spectra of buspirone hydrochloride. (A) Full-scan spectrum; (B) expanded

mass spectrum of the region around m/z 807; and (C) MS/MS fragment ion spectrum of the ion at

m/z 807.

ESI-MS of noncovalent complexes of ligand hydrochlorides 2141


Table 1. Structure of the arylpiperazine hydrochlorides studied (1–16) and nominal masses (Da) of their free bases and of the

complex ions formed.

Compound n Ar R Base formula (M)Nominal

mass (Mn)Formula of

hydrochloride

Nominal mass of[2(MþH)þþCl�]þ

complex

1 4 C21H31N5O2 385 M . HCl 807

2aa,b 3 C21H23N3O3 365 M . HCl —

2ba 4 C22H25N3O3 379 M . HCl 795

3aa,b 3 C21H22N3O3Cl 399 M . 2 HCl . 0.5 H2O —

3ba 4 C22H24N3O3Cl 413 M . HCl 863

4ba 4 C23H27N3O4 409 M . HCl . 0.5 H2O 855

5aa,b 3 C20H23N3O2 337 M . HCl —

5ba 4 C21H25N3O2 351 M . HCl 739

6ba 4 C21H24N3O2Cl 385 M . HCl 807

7aa,b 3 C21H25N3O3 367 M . HCl —

7ba 4 C22H27N3O3 381 M . HCl . 0.5 H2O 799

8bc 4 C19H23N5O2 353 M . HCl . H2O 743

Continues



9ad 3 C21H25N3O3 351 M . 2 HCl —

10ba 4 C22H26N3O2Cl 399 M . HCl (M . 2 HCl) 835

11ba 4 C23H29N3O3 395 M . HCl 827

12bc 4 C20H25N5O2 367 M . HCl 771

13ae 3 H C13H20N2 204 M . 2 HCl 445

13bf 4 H C14H22N2 218 M . HCl 473

14ae,g 3 H C13H19N2Cl 238 M . 2 HCl 513

14bf,g 4 H C14H21N2Cl 252 M . 2 HCl 541

15ah 3 H C14H22N2O 234 M . 2 HCl 505

15bh 4 H C15H24N2O 248 M . 2HCl . 0.5 H2O 533

16ai 3 H C11H18N4 206 M . 2 HCl 449

16bh 4 H C12H20N4 220 M . 2 HCl 477

a,b,d,e,f,g,h,i Data taken from Refs. 13, 16, 17, 24, 25, 26, 27 and 28, respectively. cNew compounds, see Experimental section.

Table 1. Continued

Compound n Ar R Base formula (M)Nominal

mass (Mn)Formula of

hydrochloride

Nominal mass of[2(MþH)þþCl�]þ

complex



the fact that it was recorded using an excess of 7a. Hence, it

was confirmed that the length of a spacer is indeed a key

feature influencing the complex ion formation; however, in

the presence of a proper partner, compounds of set a are also

able to form these adducts under ESI conditions.

In order to explore the role of a terminal lactam fragment on

the complex formation, additional mass spectra of hydro-

chloride salts of simplen-propyl (13a–16a) and n-butyl (13b–

16b) derivatives of 1-arylpiperazines were analyzed (Table 1).

In all these cases, peaks corresponding to the [MþH]þ ions of

the bases, and also corresponding to the respective

[2(MþH)þþCl�]þ complexes, were dominant signals. Thus,

it may be concluded that the presence of a suitable terminal

fragment, together with the spacer length, control the

association process.

Since ESI-MS experiments do not provide any direct

information about the structures of the detected complexes,

these can only be deduced on the basis of some premises. Due

to a large difference in the basicity of the piperazine nitrogen

atoms, there is no doubt that the primary protonation site is

the N4 atom, substituted with an aliphatic chain. This could

also be observed in numerous crystallographic structures of

arylpiperazine salts, in which the N4 atom was protonated

with the chloride anion located close above, within 3.0–3.1

A.19–21 Such ionic interactions also play a decisive role in

vacuum, and thus the most likely complex architecture

Figure 2. ESI mass spectra of mixtures of the hydrochlorides of buspirone and (A) 7a or (B) 7b.



should contain an NHþ��Cl��NHþ bridge. The above

reasoning was supported by an additional experiment using

triethylamine hydrochloride as a model compound in which

complex formation was possible only due to ionic forces.

Since a strong signal atm/z 239, corresponding to the complex

ion, was observed (Fig. 3), it seems that in the case of small

basic compounds ionic interactions are sufficient for adduct

formation. For larger compounds with a flexible structure,

other noncovalent forces (electrostatic, aromatic and hydro-

phobic) may play either a positive or a negative role. A bulky

terminal group in the case of compounds with a shorter

spacer may sterically disturb the ionic bridge and prevent

complex formation, whereas a longer and more flexible

aliphatic linker allows better adjustment. To test this

hypothesis, additional experiments with compounds with

different (in size and nature) terminal groups will be

necessary.

CONCLUSIONS

ESI-MS provides valuable information on structurally speci-

fic biomolecular interactions, especially for the purpose of

characterizing the noncovalent complexes of biomacromole-

cules,22 as well as small molecule associations.23 An ESI ana-

lysis of the arylpiperazine derivatives investigated earlier as

serotonin 5-HT1A receptor ligands showed that some amine

hydrochlorides were able to form [2(MþH)þþCl�]þ com-

plex ions. It has been found that the association process

depends on the structural features of ligands, probably due

to unfavorable steric interactions; shorter aliphatic linkers

and the presence of terminal amide moieties prevent complex

formation. Additional experiments with model triethylamine

hydrochloride strongly support the hypothesis that the

NHþ��Cl��NHþ bridge may determine the 3-D structure

of these complexes.

Besides some basic structural studies with the detected

complexes, it is interesting to analyze the discovered

phenomenon in a more general context. Although the

environment of the ESI process and the serotonin receptor

binding pocket are very different, both the [2(MþH)þþCl�]þ

complex formation and the ligand–receptor interactions are

driven by noncovalent forces. Moreover, in both these cases,

ionic interactions are considered to be the most important.

Although there is no other reason to suppose why the ability

of complex formation should be connected with biological

activity of arylpiperazine derivatives, it is intriguing to note

that compounds with a 4-carbon chain always display higher

affinity for 5-HT1A receptors than do their 3-carbon analo-

gues. Thus it may be of interest to further determine whether

the data obtained from ESI-MS spectra could be of any value

for ligand–receptor interaction studies.

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electrospray mass spectrometric studies of noncovalent complexes of buspirone hydrochloride and...

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