DYNO‐MILL‐Assisted Mechanochemistry: Design of Experiments Optimization for Sustainable Synthesis of Pyrrolo[3,4‐c]pyrazoles

Abstract

We report a sustainable DYNO‐MILL‐assisted mechanochemical synthesis of pyrrolo[3,4‐c]pyrazoles via (3 + 2) cycloaddition between in situ generated nitrile imines and maleimides. Design of experiments (DoE) optimization identified key milling parameters, enabling efficient product formation in up to 78% yield under solvent‐minimized, catalyst‐free conditions. The method provides broad substrate scope, operational simplicity, and scalability. Comparative green metrics analysis highlights reduced waste generation and improved sustainability relative to conventional solution‐phase approaches.

DoE‐guided horizontal bead milling (DYNO‐MILL) provides a sustainable route to pyrrolo[3,4‐c]pyrazoles through mechanochemical (3 + 2) cycloaddition of nitrile imines and maleimides. The method offers minimal solvent use, catalyst‐free conditions, broad substrate scope, and improved green metrics compared with traditional solution‐based methods.

1. Introduction

Mechanochemistry has emerged as a powerful strategy for advancing sustainable organic synthesis by enabling chemical transformations through the direct input of mechanical energy while minimizing solvent consumption and waste generation [1, 2]. Over the past decade, mechanochemical reactions performed in planetary or vibratory ball mills have demonstrated significant advantages compared to conventional solution‐based processes, including shorter reaction times, simplified work‐up procedures, and improved green chemistry metrics [3, 4, 5, 6, 7]. Despite these advances, most mechanochemical studies remain restricted to laboratory‐scale ball‐mill devices, which often suffer from limitations in energy transfer efficiency, heat dissipation, and scalability when translating reactions to larger production scales [8].

To overcome these limitations, alternative mechanochemical reactors capable of delivering higher energy input and improved process control are increasingly being investigated [9]. Among these, horizontal bead‐milling technology (DYNO‐MILL) represents a particularly promising platform [10, 11]. Originally developed for industrial wet‐milling processes such as pigment dispersion, paint and lacquer manufacturing, and disruption of biological materials, the DYNO‐MILL provides intense mechanical activation through a rapidly rotating agitator shaft equipped with specialized discs (DYNO‐ACCELERATOR) operating within a cooled grinding chamber [12, 13]. During operation, small grinding media—typically yttria‐stabilized zirconia beads—are accelerated together with the reactants, generating high‐frequency shear, impact, and shock forces that efficiently transfer mechanical energy to the reaction mixture. In addition to improved energy transfer, horizontal bead mills offer important operational advantages, including precise control over parameters such as rotor speed, bead loading, and residence time, as well as efficient heat management through an integrated cooling/heating system. These features make bead‐milling reactors attractive tools for the development of scalable and sustainable mechanochemical transformations.

In parallel with the development of greener synthetic technologies, the efficient construction of bioactive heterocyclic scaffolds remains a central objective in medicinal chemistry. Among these, pyrrolo[3,4‐c]pyrazole frameworks have attracted considerable attention owing to their presence in biologically active molecules and functional materials [14, 15]. Compounds containing this fused bicyclic core exhibit diverse biological activities, including inhibition of aurora kinases, glycine transporters, HIV‐1 integrase, and glycogen synthase kinase‐3, highlighting their significance as pharmacologically relevant scaffolds and valuable building blocks for drug discovery [16, 17, 18, 19, 20, 21]. Consequently, the development of efficient and sustainable synthetic routes to access this structural motif remains highly desirable.

One of the most powerful approaches for constructing pyrazole‐ and pyrazoline‐based heterocycles involves the 1,3‐dipolar cycloaddition of nitrile imines with suitable dipolarophiles. Nitrile imines are highly reactive 1,3‐dipoles capable of undergoing cycloaddition reactions with alkenes and alkynes to generate structurally diverse nitrogen‐containing heterocycles [22]. Owing to this versatile reactivity, nitrile imines have found widespread applications in medicinal chemistry, materials science, and bioorthogonal chemistry. Typically, these 1,3‐dipoles are generated in situ from hydrazonyl halides through base‐promoted dehydrohalogenation or through photolysis or thermolysis of tetrazole precursors [23, 24, 25, 26]. However, many of these strategies require prefunctionalized substrates, stoichiometric halogenating reagents, or specialized reaction conditions, which may reduce their practicality and sustainability.

Extensive studies have demonstrated that (3 + 2) cycloadditions of nitrile imines with electron‐deficient alkenes, such as maleimides, acrylates, and enamides, provide efficient access to functionalized pyrazolines and pyrazoles with broad substrate scope and good regioselectivity (Scheme 1a,b) [23, 24, 25, 26]. In particular, trapping nitrile imines with maleimides represents a straightforward strategy to construct pyrrolo[3,4‐c]pyrazole frameworks, which are important fused heterocycles for medicinal chemistry applications. Nevertheless, most reported methods rely on solution‐based conditions involving elevated temperatures, prolonged reaction times, or the use of oxidants and transition‐metal catalysts, which diminish the overall sustainability of the process (Scheme 1a,b) [23, 24, 25, 26]. For example, nitrile‐imine cycloadditions performed in solution frequently require temperatures up to 60–90 °C and reaction times extending from several hours to days to achieve satisfactory conversions.

SCHEME 1.

Reported strategies and present work for the synthesis of pyrrolo[3,4‐c]pyrazole derivatives. (a,b) Previously reported methods involving nitrile imine precursors or related intermediates reacting with electron‐deficient alkenes under metal‐, iodine‐catalyzed, or base‐mediated conditions, typically performed on millimolar scale. (c) This work: horizontal bead‐milling (Dyno‐Mill) enabled (3 + 2) cycloaddition between in situ generated nitrile imine precursors and maleimides, optimized through design of experiments (DoE), providing efficient access to pyrrolo[3,4‐c]pyrazole derivatives under solvent‐minimized conditions with scalability to molar scale.

Recent advances in mechanochemistry have enabled sustainable and efficient routes to complex heterocycles. For example, Jasiński and co‐workers reported a solvent‐free ball‐milling strategy for the synthesis of trifluoromethylated pyrrolo[3,4‐c]pyrazoles, where in situ generated CF₃‐nitrile imines from hydrazonoyl halides were trapped with maleimides through a (3 + 2)‐cycloaddition [27].

More broadly, mechanochemical activation has emerged as a powerful and environmentally benign platform for 1,3‐dipolar cycloaddition reactions under solvent‐free conditions [2, 28, 29, 30, 31]. This approach has been successfully applied to the synthesis of diverse heterocyclic frameworks, including triazoles, isoxazoles, and pyrazoles, often offering shorter reaction times, improved efficiencies, and distinct selectivities compared to conventional solution‐phase methods [31]. In particular, ball‐milling protocols have shown that in situ generated nitrile imines can be efficiently trapped by electron‐deficient dipolarophiles such as maleimides to rapidly access pyrrolo‐pyrazole derivatives. Despite these advances, current studies remain largely confined to conventional planetary ball mills, while the use of continuous and scalable mechanochemical reactors for such transformations is still underexplored.

In this context, we sought to investigate the potential of horizontal bead‐milling mechanochemistry using a DYNO‐MILL reactor for the sustainable synthesis of pyrrolo[3,4‐c]pyrazole derivatives. Herein, we report a mechanochemical (3 + 2) cycloaddition between in situ generated nitrile imines and maleimides performed in a horizontal bead mill under solvent‐minimized conditions (Scheme 1c). Guided by a design of experiments (DoE) optimization of key operational parameters, an efficient and scalable protocol was developed, demonstrating the potential of horizontal bead‐milling technology as a sustainable platform for the synthesis of complex heterocyclic scaffolds.

2. Results and Discussion

2.1. Preliminary Studies

The feasibility of the proposed (3 + 2) cycloaddition between the in situ generated nitrile imine precursor and maleimide was first explored under mechanochemical activation using a vibratory shaker mill (vsm) under liquid‐assisted grinding (LAG) conditions 1a:2a:3a in the ratio of 4:2:1 equivalence (refer Section S2a, Table S1). Encouragingly, milling with three balls of 5 mm at 30 Hz for 60 min significantly enhanced the reaction outcome, affording the product in 48%–51% yield depending on the LAG solvent employed (refer Table S1, entries 7–8, Table S1, Section S2a). Further improvement was achieved by increasing the milling intensity. Using two 7 mm balls at 70 Hz for 60 min provided yields of 50%–58% (refer Table S1, entries 9–10, Section S2a) and extending the milling time to 90 min increased the yield to 66% (refer Table S1, Entry 11, Section S2a). These results clearly demonstrate that mechanical activation substantially improves the efficiency of the cycloaddition compared to conventional solution‐based procedures.

For comparison, a few exploratory trials were performed under conventional solution‐phase conditions using different solvents and stoichiometries; however, these experiments afforded only low to moderate yields (15%–39%), confirming that the reaction is inefficient in solution and reinforcing the need for a mechanochemically driven approach (refer Table S1, entry 1–6, Section S2a). A range of solvents, including dichloromethane (DCM), dichloroethane (DCE), acetonitrile (CH₃CN), tetrahydrofuran (THF), ethanol (EtOH), and ethyl acetate (EtOAc), were evaluated during reaction optimization (Table S1). Among these, DCM, DCE, and EtOAc provided the best reaction outcomes. Consequently, DCM and EtOAc were used as LAG agents for the aforementioned experiments in the vibratory shaker mill.

Despite the improved performance observed under vibratory shaker milling conditions, practical limitations related to energy transfer efficiency, process control, and scalability remain. Planetary ball mills operate in batch mode and provide limited control over key process parameters, which can restrict their applicability for larger‐scale synthesis. In contrast, DYNO‐MILL horizontal bead‐milling technology enables continuous and highly efficient mechanical activation through intense shear and impact forces generated by rapidly circulating grinding media. Moreover, the system allows precise control over operational parameters such as rotor speed, bead loading, and residence time, while the integrated cooling system facilitates efficient temperature management during high‐energy milling and/or exothermic chemical transformation. Based on these advantages, horizontal bead milling was selected as the mechanochemical platform for further reaction optimization.

2.2. DOE

To systematically identify optimal reaction conditions, a DoE approach was subsequently employed to evaluate the influence of key process variables, including rotor speed, bead size, bead filling ratio, residence time, and LAG conditions, ultimately enabling the development of a robust and scalable protocol for the synthesis of pyrrolo[3,4‐c]pyrazole derivatives. In a first time, seven variables with fixed range were defined such as reaction time (30–60 min), rotor speed (1500–6000 rpm), equivalent of 1a (1–4 eq.), equivalent of 2a (1–2 eq.), equivalent 3a was fixed as the limiting reagent, beads filling rate (50%–70%), types of bead (ZrSiO₄ or ZrO₂/Y₂O₃), and the choice of solvent (EtOAc, DCM, or neat) as LAG in a window from 0.5 to 2.0 mL/g to obtain molecule 4a (Scheme 2). A D‐optimal design was selected to distribute experiments in the chemical space. Preliminary results showed that a high rotor speed of 6000 rpm tends to give lower yield, so a maximum speed of 4000 rpm was set to refine the experimental dataset.

SCHEME 2.

DoE‐guided optimization of the mechanochemical synthesis with horizontal bead‐milling (Dyno‐Mill) of 4a. A multivariate screening approach was employed to evaluate the influence of key reaction parameters (milling frequency, type of beads + filling rate, LAG, reaction time, and stoichiometry) on the (3 + 2) cycloaddition between the depicted precursors (1a–3a). The MODDE v.13.0.2.34314 program from Sartorius was used to perform the DoE and the optimization [32].

Three types of responses were measured, yield (left plot, Figure 1), space‐time‐yield (STY, middle plot, Figure 1), and real‐atom‐economy (RAE, right plot, Figure 1) to assess efficiency, productivity, and greenness of the methodology in order to optimize the overall reaction. Mathematical modeling shows good trend with fitting values (R ²) above 0.8 for all the corresponding responses and prediction values (Q ²) above 0.5. Those results support the soundness and predictive capability of the established model. Furthermore, the value of the model validity being greater than 0.25 indicates that there is no problem with potential outliers or transformation and the reproducibility being greater than 0.5 guarantees a correct model.

FIGURE 1.

Mathematical modeling answers toward predictive capability, validity, and correctness of the model.

Looking at the answers of the various parameters through contour plot, we can observe it is necessary to have high equivalence of 1a given that higher responses are obtained for equivalent levels above 2.5 (Figure 2A–C). Even the RAE required around 3 eq. of 1a before dropping down which indicates that excess is required to increase produced amount of 4a as well as to generate less waste (Figure 2C). In contrast, even if the increase of the number of equivalents of 2a gives better yield, the RAE drops down (Figure 2A,C). Especially for this reason, a compromise must be made between equivalents of 1a and 2a. Paying close attention to the STY, an optimal emerges at around 3.6 eq. of 1a and 1.6 eq. of 2a, respectively (Figure 2B). Rotation speed and reaction time were parameters that provided the most important effect for the yield and the STY (Figure 3A,B). Higher rotation speed indicates that too much energy is transferred by the DYNO‐MILL providing higher amount of undesired by‐product, potentially from decomposed aryl diazonium salts (through N₂↑ evolution from 2) and diazo dimers from 1. Due to the complex nature of the reaction mixture, it was difficult to isolate these by‐products for characterization. Reversely, the lowest rotation speed of the DYNO‐ACCELERATOR (1500 rpm) was defined as the ideal rotation speed given access to highest yields of 4a (Figure 3A). On the same plot, we can observe that it is crucial to keep the reaction time above 65 min to achieve a yield higher than 70% but also to avoid penalizing STY too significantly. Regarding the various solvents screened within the framework of the DOE, it appears that DCM shown to be the best LAG performance. A higher LAG value provides better yield but strongly decreases the greenness of the methodology, that is, the RAE. As a compromise between yield and green metrics, a value between 0.5 and 1 mL/g should be selected as optimal (Figure 3B). Finally, it was interesting to observe that the types of beads and the filling rate of beads do not significantly influence the outcome (refer Figures S3–S5).

FIGURE 2.

(A) Contour plot of yield [%], (B) space‐time‐yield [g/L/h], and (C) real‐atom economy [%] between the eq. 1a and eq. 2a. Reaction parameters were fixed as follows: reaction time: 65 min, rotor speed: 1500 rpm, LAG: 1 ml/g of DCM, beads filling rate: 60% with ZrO₂/Y₂O₃.

FIGURE 3.

(A) Contour plot of the yield [%] between rotor speed [rpm] and the reaction time [min]. Reaction parameters were fixed as follows: eq. 1a: 3.6, eq. 2a: 1.6, LAG: 1 ml/g of DCM, beads filling rate: 60% with ZrO₂/Y₂O₃. (B) Contour plot of the yield [%] between the amount of LAG [mL/g] and the eq. 1a. Reaction parameters were fixed as follows: reaction time: 65 min, rotor speed: 1500 rpm, eq. 2a: 1.6, solvent: DCM, beads filling rate: 60% with ZrO₂/Y₂O₃.

Given their comparable performance, EtOAc was selected as the preferred LAG solvent for the final library synthesis because of its lower environmental impact and the potential volatility‐related limitations of DCM under milling conditions. It is noteworthy that continuous cooling was applied throughout the process, although the internal reaction temperature could not be directly controlled. No solvent replenishment was required during optimization.

2.3. Library Synthesis Using the Optimized Horizontal Bead‐Milling Protocol

With the optimized conditions in hand, the generality of the Dyno‐Mill‐assisted mechanochemical protocol was investigated through the synthesis of a diverse library of pyrrolo[3,4‐c]pyrazole derivatives (Scheme 3). The reactions were performed using 0.5 mm ZrO₂/Y₂O₃ beads with 60% bead filling at 1500 rpm under liquid‐assisted grinding conditions (η = 0.5, ethyl acetate) with numerous ethyl and methyl diazoacetates 1a/ 1b, aryl diazonium tetrafluoroborates 2 and NH/N‐substituted maleimides 3 (Scheme 2) in a 80 mL reaction chamber. Under these optimized parameters, a broad range of aryl diazonium‐derived nitrile imine precursors and maleimide dipolarophiles underwent efficient (3 + 2) cycloaddition to afford the desired bicyclic products in moderate to excellent yields (59%–90%), demonstrating the robustness of the DYNO‐MILL horizontal bead‐milling protocol as well as the outcome of the DoE study.

SCHEME 3.

Substrate scope for the DYNO‐MILL‐enabled mechanochemical synthesis of pyrrolo[3,4‐c]pyrazole derivatives via (3 + 2) cycloaddition of in situ generated nitrile imines with N‐substituted maleimides. Reactions were performed using ZrO₂/Y₂O₃ beads (0.5 mm, 60% bead filling, 90 g) at 1500 rpm under liquid‐assisted grinding conditions (η = 1.0, DCM).

The substrate scope first examined variations in the aryl component of the nitrile imine precursor (2a–2k). A range of electron‐withdrawing substituents on the aromatic ring were well tolerated under the milling conditions. Substrates bearing trifluoromethyl groups (2a, 2d, and 2e) provided products such as 4a (71%), 4d (65%), 4e (74%), 4i (70%), 4o (70%), 4p (71%), 4x (70%), 4ad (70%), and 4af (75%) in good yields (Scheme 3). Similarly, halogenated aryl derivatives containing bromo, chloro, fluoro, and iodo substituents (2c, 2f, 2g, 2h, and 2i) were efficiently converted to the corresponding cycloadducts (Scheme 3). For example, brominated substrates (2f) delivered 4f (84%) and 4r (88%), while chloro‐substituted analogs (2g and 2i) produced 4g (80%), 4j (65%), 4n (80%), 4s (80%), and 4ah (88%). Fluoro‐containing derivatives (2h) were also well accommodated, affording 4l (73%), 4v (65%), and 4ab (60%). Notably, iodo‐substituted aryl precursors (2c) yielded products such as 4c (85%), 4h (68%), 4m (87%), and 4w (85%), demonstrating that even sterically demanding and potentially reactive halogens remain intact under the mechanochemical conditions. The tolerance of halogenated arenes is particularly advantageous as these substituents serve as useful synthetic handles for subsequent cross‐coupling transformations (Scheme 3).

The method also displayed good compatibility with electron‐donating substituents and sterically demanding groups on the aryl ring. For instance, substrates bearing alkyl substituents (2k) afforded products such as 4t (81%), while the presence of a bulky tert‐butyl substituent (2j) delivered 4q in an excellent 90% yield, representing the highest yield within the library (Scheme 3). These results indicate that steric hindrance on the aromatic ring has minimal influence on the efficiency of the cycloaddition under the DYNO‐MILL conditions.

Next, the influence of the maleimide dipolarophile (3a –3e) was examined. Both N‐benzyl and N‐phenyl maleimides 3a and 3b, respectively, were compatible with the optimized milling protocol, enabling the formation of structurally diverse fused heterocycles. 3b generated products such as 4h (68%), 4i (70%), and 4j (65%), whereas N‐alkyl maleimides 3c and 3d produced derivatives including 4k (75%), 4l (73%), 4m (87%), 4n (80%), 4o (70%), 4p (71%), 4q (90%), and 4r (88%), respectively (Scheme 3). These results demonstrate that variations in the nitrogen substituent of the maleimide have only a modest influence on reaction efficiency.

The protocol also tolerated variations in the ester functionality present on the nitrile imine precursor, including both ethyl and methyl ester groups 1a and 1b, respectively, which remained intact throughout the mechanochemical process. Products such as 4c (85%), 4j (65%), 4k (75%), and 4y (85%) illustrate that the ester moiety is preserved under the milling conditions, providing convenient sites for further synthetic modification or derivatization (Scheme 3).

Finally, substrates containing free imide NH functionalities were also successfully synthesized from 3e, affording compounds such as 4aa (80%), 4ab (60%), 4ac (82%), 4ad (70%), 4ae (66%), 4af (75%), 4ag (85%), 4ah (88%), and 4ai (75%), indicating that the optimized mechanochemical conditions tolerate both protected and unprotected imide motifs (Scheme 3).

Overall, the substrate scope demonstrates the broad functional group compatibility of the DYNO‐MILL protocol. A wide range of substituents—including halogens (F, Cl, Br, I), trifluoromethyl groups, alkyl substituents, tert‐butyl groups, ester functionalities, and diverse N‐substituted maleimides—are tolerated without significant loss in efficiency. The reactions consistently delivered the desired products in moderate to excellent yields (59%–90%), underscoring the efficiency and robustness of the DYNO‐MILL ‐assisted mechanochemical process. It is noteworthy that the electronic nature of 2 strongly influenced the reaction outcome. Substrate 2j bearing an electron‐donating tert‐butyl group delivered the highest yield (90%), likely due to enhanced stabilization and reactivity of the transient nitrile imine intermediate. Electron‐donating substituents increase the HOMO energy of the dipole, facilitating cycloaddition with the electron‐deficient dipolarophile and suppressing competing decomposition pathways. In contrast, electron‐withdrawing groups such as trifluoromethyl and esters reduced the reaction efficiency, presumably by lowering the dipole HOMO and slowing the cycloaddition process. Highly electron‐rich aryl diazonium salts were not investigated, as these substrates are often unstable under mechanochemical milling conditions.

To demonstrate the scalability of the mechanochemical protocol, the model reaction was performed on a multigram scale using 2.0 g of N‐benzylmaleimide (3a). With CH₂Cl₂ as LAG (0.5 mL/g) at 1500 rpm for 4 h, the desired product was obtained in moderate yield. Although the conditions remain unoptimized, the result highlights the potential of this methodology for larger‐scale mechanochemical synthesis.

These results highlight the utility of horizontal bead milling tools, like DYNO‐MILL, as a powerful and scalable platform for the rapid synthesis of structurally diverse pyrrolo[3,4‐c]pyrazole derivatives, providing access to a library of biologically relevant heterocycles with high functional group tolerance under solvent‐minimized mechanochemical conditions.

To elucidate the underlying reaction pathway, preliminary control expeiments were performed (Scheme 4A). When the model reaction of 1a, 2a, and 3a was subjected to the optimized conditions in the presence of radical scavengers TEMPO or BHT (2 eq.), the formation of product 4a was largely unhindered. The desired product was isolated in 67% and 70% yields, respectively. The lack of significant reaction suppression by these radical traps strongly suggests that the transformation proceeds via an ionic mechanism rather than a radical‐mediated sequence. Interestingly, the same reaction under solution required nearly 10 h to complete and provide 4a in comparable yields.

SCHEME 4.

(A) Control experiment to elucidate the mechanism of pyrrolo[3,4‐c]pyrazole. (B) Putative mechanism for the formation of 4.

Mechanistically, the reaction is driven by the in situ generation of a highly reactive nitrile imine intermediate E (Scheme 4B). This forms via the initial coupling of alkyl diazoacetate 1 and aryl diazonium tetrafluoroborate 2 to yield intermediate A. Subsequent extrusion of nitrogen gas (N₂↑) provides intermediate B, which then undergoes a 1,3‐hydride shift to generate C, and then subsequent elimination of HBF₄ from C produced the nitrile imine D/E. Finally, a standard (3 + 2) cycloaddition between this intermediate and the N‐substituted maleimide 3 affords the final pyrrolo[3,4‐c]pyrazole scaffold 4.

2.4. Green Metrics Analysis

To quantitatively evaluate the sustainability of the developed mechanochemical protocol, key green chemistry metrics including atom economy (AE), process mass intensity (PMI), and E‐factor were calculated and compared with two representative literature procedures [25, 33, 34]. The corresponding reaction strategies are illustrated in Scheme 5a–c, while the comparative analysis of the green metrics is presented as a bar plot in Scheme 5d and in the Supporting Information (refer Section S4). Specifically, the mechanochemical DYNO‐MILL protocol (Scheme 5c) is compared with a representative solution‐phase method (Scheme 5b) and a CuCl‐catalyzed strategy (Scheme 5a) [25, 34].

SCHEME 5.

Comparison of green metrics from Zhao et al. (a), Benassi et al. (b), and the Dyno‐Mill (c). (Scheme 3a) Protocols using actual values. (d) Atom economy (AE), process mass intensity (PMI), E‐factor, and EcoScale are shown on a logarithmic scale to account for the large differences in material efficiency. The Zhao et al. method exhibits significantly higher PMI and E‐factor values, reflecting its solvent‐intensive nature. We deliberately limited the green metrics evaluation to the chemical transformation step, excluding downstream operations due to insufficient detail in the available supporting information to enable a reliable and quantitative assessment of solvent and reagent consumption.

As shown in the bar plot in Scheme 5d, the AE values of the three strategies are broadly comparable, reflecting the inherent efficiency of the (3 + 2) cycloaddition approach used to construct the pyrrolo[3,4‐c]pyrazole scaffold. The protocol reported by Benassi and co‐workers (Scheme 5b) [34] and the Zhao method (Scheme 5a) [25] display slightly higher AE values, while the present DYNO‐MILL strategy (Scheme 5c) remains competitive. This confirms that the intrinsic reaction design across all three methods efficiently incorporates a large proportion of the reactant atoms into the desired heterocyclic framework.

More pronounced differences are observed when evaluating material efficiency metrics, particularly PMI and E‐factor. As summarized in the Supporting Information (refer Section S4) and reflected in Scheme 5d, the two literature methods exhibit significantly higher material consumption and waste generation due to their reliance on bulk solvents, additional reagents, and energy‐intensive conditions. Notably, the Zhao protocol (Scheme 5a) displays exceptionally high PMI and E‐factor values (1078 and 1077, respectively) (refer Section S4), which are orders of magnitude greater than those observed for both the DYNO‐MILL and Benassi et al. protocols. These elevated values arise primarily from the use of large volumes of DMF, the presence of CuCl catalyst, and solvent‐intensive purification steps, whereby reaction and work‐up solvents dominate the overall material balance. Similarly, the Benassi protocol (Scheme 5b) requires triethylamine and high‐boiling m‐xylene under microwave heating (150 °C), contributing to increased material input and waste generation, albeit to a lesser extent than the Zhao method.

In contrast, the DYNO‐MILL mechanochemical strategy (Scheme 5c) simplifies the reaction medium and reduces several sources of waste. The reaction proceeds under LAG conditions using only a minimal amount of dichloromethane while avoiding both transition‐metal catalysts and stoichiometric additives. Although the PMI and E‐factor values appear closer than expected at laboratory scale, this arises from the inclusion of downstream processing materials (e.g., extraction and chromatographic solvents) in the calculations, which can dominate the overall material balance. Consequently, these metrics tend to underestimate the intrinsic advantage of solvent‐minimized mechanochemical processes. Importantly, the DYNO‐MILL protocol eliminates catalyst‐derived waste and significantly reduces solvent dependency during the reaction step, leading to an overall improved sustainability profile.

Another important advantage illustrated in Scheme 5c is the process simplification enabled by mechanochemistry. Conventional solution‐phase strategies (Scheme 5a,b) require elevated temperatures (80–150 °C), specialized reaction conditions, and longer reaction times, whereas the DYNO‐MILL protocol achieves efficient product formation through mechanical activation at ambient temperature and shorter reaction times. The enhanced energy transfer provided by the horizontal bead‐milling reactor promotes rapid generation of the nitrile imine intermediate and its subsequent cycloaddition with the maleimide dipolarophile, thereby improving the operational efficiency of the synthesis. In addition, the literature procedures require heating and subsequent cooling of the reaction medium, operations that significantly increase the overall energy demand. In contrast, the DYNO‐MILL protocol operates under mild conditions without external heating and would therefore be expected to exhibit a substantially improved sustainability profile when evaluated through a full life‐cycle assessment (LCA) (refer Section S4).

EcoScale analysis (Scheme 5d) further supports these observations. Based on experimental data, the DYNO‐MILL protocol achieves an EcoScale score of 50, compared to 43 for the Benassi protocol and 33 for the Zhao method. The higher score of the mechanochemical strategy primarily arises from the absence of transition‐metal catalysts, reduced solvent usage, and operation under mild temperature conditions, whereas penalties in all cases are largely associated with chromatographic purification.

Overall, the comparative analysis summarized in Scheme 5 highlights the superior sustainability profile of the DYNO‐MILL method, which combines competitive AE with significantly reduced material input, lower waste generation, simplified processing, and reduced energy demand. These features underscore the potential of horizontal bead‐milling mechanochemistry as a green‐by‐design synthetic platform, providing a viable and scalable alternative to traditional solution‐phase methodologies for the construction of complex heterocyclic frameworks.

3. Conclusion

In summary, we have developed a sustainable and scalable mechanochemical strategy for the synthesis of pyrrolo[3,4‐c]pyrazole derivatives via a (3 + 2) cycloaddition between in situ generated nitrile imines and maleimides using horizontal bead‐milling technology (DYNO‐MILL). Preliminary investigations under conventional solution‐phase conditions provided only modest efficiencies, whereas mechanochemical activation significantly enhanced the reaction outcome through improved mixing and energy transfer. Guided by a DoE strategy, key operational parameters—including rotor speed, bead size, bead filling ratio, residence time, and LAG conditions—were systematically optimized to establish a robust and scalable protocol.

The optimized methodology enabled the synthesis of a diverse library of pyrrolo[3,4‐c]pyrazole derivatives in moderate to excellent yields (59%–90%), demonstrating broad tolerance toward electron‐withdrawing and electron‐donating substituents, halogens, trifluoromethyl groups, alkyl substituents, ester functionalities, and various N‐substituted maleimides. Importantly, the process operates under solvent‐minimized conditions, avoids transition‐metal catalysts, and proceeds without external heating, highlighting its operational simplicity and functional group compatibility for the rapid generation of heterocyclic libraries.

A comparative green metrics analysis further underscores the sustainability advantages of the developed protocol. Evaluation of parameters such as AE, PMI, EcoScale, and E‐factor in comparison with representative literature procedures revealed that the DYNO‐MILL strategy significantly reduces material input and waste generation. In particular, the markedly lower PMI and E‐factor values relative to solvent‐intensive protocols highlight the benefits of minimizing solvent usage and eliminating catalyst‐derived waste while maintaining competitive AE. The mechanochemical process also benefits from reduced reaction times, simplified reaction media, and improved energy efficiency, collectively contributing to a more environmentally benign synthetic route.

Overall, this work demonstrates that horizontal bead‐milling mechanochemistry provides a powerful green‐by‐design platform for complex heterocycle synthesis. The combination of continuous high‐energy milling and DoE‐guided optimization offers a promising strategy for translating mechanochemical transformations toward practical, scalable, and industrially relevant synthetic processes.

Funding

This study was supported by Shiv Nadar University.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

The supporting information is available free of charge at https://doi.org/10.1002/cssc.xxxxxxxx. Experimental procedures, green metrics, ¹H, ¹³C NMR, and spectra data of compounds.

Pandey Roopam, Mariaux Rémy, Bera Subhankar, Verma Khushi, Gremaud Ludovic, Sen Subhabrata, DYNO‐MILL‐Assisted Mechanochemistry: Design of Experiments Optimization for Sustainable Synthesis of Pyrrolo[3,4‐c]pyrazoles, ChemSusChem 2026, 19, e70888. 10.1002/cssc.70888

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.