Abstract
Four fused nitrogen‐containing ring systems were investigated by electrospray ionization–tandem mass spectrometry: Pyridazino‐indoles, pyridazino‐quinolines, a pyrimido‐quinoline derivative and pyrimido‐cinnolines. Fragmentation patterns of these compounds are discussed and compared. Several characteristic cross‐ring fragments were formed mainly on the pyridazine and pyrimidine rings of the ring systems. The connected Cl, NO2, Me, Ph and more extended heterocyclic substituents influenced the fragmentation.
Keywords: fragmentation, fused N‐heterocycles, pyridazino‐indoles, pyridazino‐quinolines, pyrimido‐cinnolines, pyrimido‐quinolines, tandem mass spectrometry
1. INTRODUCTION
N‐containing heterocyclic compounds possess diverse biological activities and frequently present in molecules of interest in medicinal chemistry. 1 , 2 , 3 Among them, the investigated ring‐systems show also several important activities: Pyridazino‐indole heterocycle is a structural element in antihypertensive, platelet‐aggregation inhibitor compounds or benzodiazepine‐receptor ligands. 4 , 5 , 6 , 7 Pyridazino‐quinolines have topoisomerase‐inhibitor and cytotoxic activites. 8 Pyrimido‐quinolines are representatives of antioxidant and antimicrobial compounds. 9 , 10 Pyrimido‐cinnolines are potent candidates for antimicrobial, anti‐inflammatory and antiplatelet application. 11 , 12
The studied compounds were synthesized by Suzuki—condensation ring closure tandem reactions as we described in our previous studies. 13 , 14 , 15 , 16 In continuation of our work on the mass fragmentation mechanisms of the thieno[3′,2′:4,5]pyrido[2,3‐d]pyridazine ring system, 17 a detailed study on fused N‐heterocycles was performed to understand the fragmentation behaviour of these compounds. This paper reports a study on the fragmentation mechanisms under ESI/MS conditions of pyridazino‐indoles (1–6), pyridazino‐quinolines (7–12), a pyrimido‐quinoline derivative (13) and pyrimido‐cinnolines (14–15). The [M + H]+ values of the compounds are summarized in Table 1.
TABLE 1.
[M + H]+ values of 1–15
| Compound | [M + H]+ |
|---|---|
| 1 | 200 |
| 2 | 262 |
| 3 | 245 |
| 4 | 279 |
| 5 | 229 |
| 6 | 263 |
| 7 | 303 |
| 8 | 337 |
| 9 | 365 |
| 10 | 411 |
| 11 | 479 |
| 12 | 535 |
| 13 | 333 |
| 14 | 243 |
| 15 | 277 |
2. RESULTS AND DISCUSSION
2.1. Pyridazino‐indoles
For 1, three peaks were detected in the MS/MS spectrum: m/z = 116, 143 and 169. The loss of pyridazine N and CH3 (as a methylamine) resulted in the fragment ion at m/z = 169. Two further cross‐ring fragments by the cross‐ring‐cleavage of the pyridazine ring were identified at m/z = 116 and 143. The fragmentation pattern of 2 showed the same m/z = 143 and m/z = 169 (m/z = 169: due to the loss of an aniline) fragments as in 1. An additional fragment at m/z = 104 is appeared due to fragmentation on the pyridazino‐indole ring system. m/z = 234 is detected as a loss of CO.
In case of 3 and 4, the chlorine on the C ring did not influence the fragmentation behaviour. A loss of the nitro group resulted in a fragment ion of m/z = 199/233. Cross‐ring fragmentation on the pyridazino ring led to m/z = 144 in 3 and m/z = 178 in 4. Similarly to 1, the loss of methylamine could be detected in the case of these compounds (m/z = 169 in 3 and m/z = 203 in 4).
Comparing 5 and 6, a more pronounced fragmentation occurred in the chlorine‐derivative 6. The loss of the nitro group was seen in both cases as in 3 and 4 (m/z = 183 in 5 and 217 in 6). Cross‐ring cleavages on the pyridazine ring generated the ions at m/z = 129 and 156 in 5, and m/z = 163 and 190 in 6. The formation of m/z = 190 was followed by a loss of the chlorine (m/z = 155) (Figure 1).
FIGURE 1.
Fragmentation schemes of 1–6
2.2. Pyridazino‐quinolines
Compound 7 showed a complex fragmentation with several cross‐ring fragments and rupture between the quinoline and the phenylamino substituent (ion of aniline at m/z = 93 and after the loss of phenyl group from the [M + H]+, the remaining cation at m/z = 226). In 8, the chlorine‐derivative of 7, similar fragmentation was observed. Cleavage of the phenylamino/phenyl moiety was also detectable: m/z = 93 and 244. In both compounds, the methylamine loss was detectable at m/z = 272 in 7 and m/z = 308 in 8. In the N‐2‐phenyl derivative (9), different fragmentation occurred in the pyridazino‐quinoline ring system, giving the fragment ions at m/z = 320 and 244. Between the phenylamino substituent and quinoline ring, the same rupture was seen as in 8 (m/z = 272). m/z = 217 could be explained as a formation of an aryne‐type cation from the quinoline. Characteristic m/z = 77 appeared from the cleavage of the phenyl moiety.
We compared compounds 7–9 with their analogue structures with an extended substituent on the quinoline ring. Rather different fragmentation patterns were observed for 10–12. These three compounds tended to produce fragment ions originated from the rupture on the pyridazine ring connected to the phenylamino moiety. The loss of methylhydrazine and phenylhydrazine resulted in the fragment ions at m/z = 367 (10), 435 (11) and 430 (12). In case of 10 and 12, no fragmentation occurred on the pyridazino‐quinoline ring system. The characteristic m/z = 77 appeared by the cleavage of the phenyl from pyridazino‐quinoline or pyridazine N‐2 in 12. In 10 and 11, two methyl groups with the formation of methane provides to peaks m/z = 380 (10) and 448 (11) (Figure 2).
FIGURE 2.
Fragmentation schemes of 7–12
2.3. Pyrimido‐quinoline
13 showed fragmentation only on the pyrimidine ring during the MS experiments. m/z = 276 fragment was detected after the ejection of a H3CNC=O. The formyl loss from this fragment was possible; resulted in m/z = 246. The quinoline ring system could remain in the fragmentation process (m/z = 220) (Figure 3).
FIGURE 3.
Fragmentation schemes of 13–15
2.4. Pyrimido‐cinnolines
14 and 15 could also fragment via the same bond cleavages as 13 on the pyrimidine ring to produce the detected ions. The cinnoline part appeared at m/z = 130 (14) and 164 (15). The cross‐ring fragment ions were m/z = 186/220; and after formyl loss 158/192 in 14/15 (Figure 3).
3. EXPERIMENTAL
3.1. General
Chemicals were obtained from VWR chemicals (France). All solvents were HPLC grade. All compounds were synthesized at the Department of Organic Chemistry, Semmelweis University, which were published previously. 13 , 14 , 15 , 16
3.2. MS and MS/MS measurements
Agilent 6460 QQQ mass spectrometer with Jet Stream electrospray ion source (ESI/MS) (Waldbronn, Germany) was used to perform MS and MS/MS measurements in positive mode. Samples were dissolved in acetonitrile: Water containing 0.5% formic acid = 1:1, 5 μl samples were injected, flow rate was 0.5 ml/min. Eluent contained acetonitrile: water containing 0.5% formic acid = 1:1.
Capillary voltage was 3500 V, the capillary temperature was kept at 300°C. Quadrupole scanned over the range m/z 50–1000 in MS measurements. In MS/MS measurements product ion scan mode was applied, protonated molecular ions (MH+) were studied. Nebulizer, sheath and collision gas was nitrogen. The set parameters were as follows: Fragment or voltage 135 V, gas flow rate 12 L/min, nebulizer gas flow 45 psi, sheath gas flow 11 L/min, sheath gas temperature (heater) 400°C. The set collision energies are summarized in Table 2. Spectra were evaluated with Agilent MassHunter B 02.01. software.
TABLE 2.
Collision energies used in tandem mass spectrometry experiments
| Compound investigated | Collision energy (eV) |
|---|---|
| 1 | 20 |
| 2 | 20 |
| 3 | 15 |
| 4 | 15 |
| 5 | 20 |
| 6 | 20 |
| 7 | 50 |
| 8 | 40 |
| 9 | 40 |
| 10 | 30 |
| 11 | 30 |
| 12 | 30 |
| 13 | 30 |
| 14 | 25 |
| 15 | 20 |
3.3. Conclusions
Fifteen nitrogen‐containing ring systems were studied by tandem mass spectrometry. In case of the six pyridazino‐indoles, all compounds showed cross‐ring fragmentation on the pyridazine ring. For the nitro derivatives, as 3–6, the loss of NO2 was also seen. In the chlorine‐containing 4 no additional fragments appeared compared to its analogue structure 3. Contrary to this, the fragmentation of the chlorine‐derivative 6 resulted in a new fragment affected by the chlorine‐substituent. Intensive fragmentation on the whole heterocylic ringsystem was detected for pyridazino‐quinolines 7–9. The other set of pyridazino‐quinolines 10–12 with an extended substituent showed fragmentation mainly on the pyridazino substituent connected to the phenylamino moiety. The chlorine‐substituent in 8 and 11 had no effect on the fragmentation. Pyrimido‐quionoline (13) and pyrimido‐cinnolines (14 and 15) tended to give fragments originated predominantly from the pyrimidine ring.
Supporting information
Figure S1: CID mass spectrum of 1
Figure S2: CID mass spectrum of 2
Figure S3: CID mass spectrum of 3
Figure S4: CID mass spectrum of 4
Figure S5: CID mass spectrum of 5
Figure S6: CID mass spectrum of 6
Figure S7: CID mass spectrum of 7
Figure S8: CID mass spectrum of 8
Figure S9: CID mass spectrum of 9
Figure S10: CID mass spectrum of 10
Figure S11: CID mass spectrum of 11
Figure S12: CID mass spectrum of 12
Figure S13: CID mass spectrum of 13
Figure S14: CID mass spectrum of 14
Figure S15: CID mass spectrum of 15
Krajsovszky G, Dalmadiné Kiss B, Ludányi K, Mándity IM, Bogdán D. Electrospray ionization–tandem mass spectrometric study of fused nitrogen‐containing ring systems. J Mass Spectrom. 2022;57(6):e4870. doi: 10.1002/jms.4870
DATA AVAILABILITY STATEMENT
Data available on request from the authors.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Figure S1: CID mass spectrum of 1
Figure S2: CID mass spectrum of 2
Figure S3: CID mass spectrum of 3
Figure S4: CID mass spectrum of 4
Figure S5: CID mass spectrum of 5
Figure S6: CID mass spectrum of 6
Figure S7: CID mass spectrum of 7
Figure S8: CID mass spectrum of 8
Figure S9: CID mass spectrum of 9
Figure S10: CID mass spectrum of 10
Figure S11: CID mass spectrum of 11
Figure S12: CID mass spectrum of 12
Figure S13: CID mass spectrum of 13
Figure S14: CID mass spectrum of 14
Figure S15: CID mass spectrum of 15
Data Availability Statement
Data available on request from the authors.