1 What Three-Dimensional Drug Scaffolds Truly Lack

1.1 Medicinal-Chemistry Value of Rigid Three-Dimensional Structures

Azabicyclo[x.1.1]alkanes (ABCAs) are a class of nitrogen-containing bridged-ring compounds. Compared with planar structures such as benzene rings and conformationally flexible nitrogen-containing heterocycles such as piperazines and diazepanes, these molecules possess greater rigidity and more clearly defined three-dimensional geometries.

Bridging constrains ring conformations and directs substituents attached to the scaffold along relatively well-defined vectors. Such geometric constraints may influence how a molecule fits into a protein-binding site and may also alter properties such as basicity, lipophilicity, solubility, and metabolic stability. Accordingly, structures such as 3,6-diazabicyclo[3.1.1]heptanes have been used as replacements for piperazines, diazepanes, and other commonly used nitrogen-containing heterocycles.

However, rigidity or three-dimensionality alone does not necessarily result in higher potency, improved selectivity, or more favorable pharmacokinetic properties. The actual effect depends on whether the spatial shape of the bridged ring is compatible with a specific target and whether the substituents are directed toward the appropriate regions of the binding site. Medicinal chemistry therefore requires more than merely “synthetically accessible three-dimensional scaffolds”; it requires three-dimensional scaffolds that can be further modified at strategically important positions.

1.2 Core Limitations of Conventional Synthetic Methods

Conventional bridged aza scaffolds are commonly constructed from monocyclic precursors through intramolecular nucleophilic substitution, C—H amination, or other ring-closing reactions. Although these methods can form bridged rings efficiently, substituents at the bridge position generally need to be installed before cyclization.

This creates three problems:

1. Each change to the bridge-position substituent usually requires the preparation of a new precursor;

2. A newly introduced substituent may affect the yield and selectivity of the subsequent ring-closing step;

3. Once the bridged ring has formed, its compact internal structure makes selective functionalization at a specific bridge position difficult.

This article is based primarily on the preprint study entitled “Modular Assembly of Bioisosteric Bridged Aza-frameworks via Strained Ring Release,” conducted collaboratively by Xiangyang Chen’s team at Shanghai Jiao Tong University and researchers at Nanjing University of Chinese Medicine. The study was posted on Research Square on October 30, 2025, with the DOI 10.21203/rs.3.rs-7615919/v1. At the time of writing, the work remains a preprint that has not yet completed peer review, and its mechanistic interpretations and scope of application therefore require further validation in subsequent studies.

2 Synthetic Design: Constructing a Common Tricyclic Precursor Before Releasing Strain

2.1 Why Azatricycloalkanes Were Selected

Rather than synthesizing each target bridged ring separately from differently substituted monocyclic starting materials, the researchers first prepared a class of azatricycloalkanes containing highly strained C—N bonds. Relative to the target azabicyclic products, these precursors contain an additional three-membered ring. The three-membered ring forces the bond angles to deviate substantially from the preferred geometries of conventional tetrahedral carbon and amine nitrogen atoms, thereby generating C—N bonds with elevated reactivity.

This structural design simultaneously defines three features:

① Target scaffold: cleavage of the tricyclic structure retains the core azabicyclo[x.1.1]alkane framework;

② Reaction site: the C—N bond designated for cleavage predetermines the bridge carbon at which the new functional group will be introduced;

③ Reaction driving force: opening of the three-membered ring releases part of the ring strain, favoring formation of a new C—X bond.

2.2 How the Computational Results Should Be Interpreted

The researchers used density functional theory (DFT) to evaluate the stability of the azatricycloalkanes and the feasibility of strain-release reactions involving their C—N bonds. The calculations showed that the bond dissociation energies (BDEs) of the relevant C—N bonds in the target azatricycloalkanes were approximately 45–57 kcal/mol. These values indicate that the C—N bonds are not exceptionally fragile and that the tricyclic precursors possess a meaningful degree of structural stability.

The researchers further used the reaction between 1-azatricyclo[4.1.0.0²,⁷]heptane and p-toluenesulfonyl chloride (TsCl) as a model. Along the calculated reaction pathway for this TsCl model system, the highest-energy step was formation of the N—S bond between the nitrogen atom and TsCl, with a Gibbs free-energy barrier of 20.6 kcal/mol. By comparison, the corresponding barrier for 1-azabicyclo[1.1.0]butane was 23.1 kcal/mol.

C—N bond cleavage and C—Cl bond formation then occur, accompanied by release of strain from the three-membered ring. These calculations support the conclusion that the tricyclic precursors possess a certain degree of stability in their unactivated state, while activation with TsCl enables selective ring opening and bridge-position functionalization through a pathway with a relatively low energy barrier.

It should be noted that BDE reflects the energy required for homolytic cleavage of a chemical bond and cannot be directly equated with ring strain. The practical utility of the tricyclic precursors must therefore be assessed by considering bond dissociation energies, reaction barriers, and experimental stability together.

2.3 How the Tricyclic Precursors Were Prepared

The researchers used a tetrahydropyridinol bearing a tert-butyloxycarbonyl (Boc) protecting group as the starting material. Sequential epoxidation, azide-mediated epoxide opening, hydroxyl-group activation, and consecutive intramolecular cyclizations were then used to construct azatricyclic precursors containing highly strained C—N bonds.

The principal synthetic sequence is shown below:

N-Boc-1,2,3,6-tetrahydropyridin-3-ol 6

                  ↓

mCPBA, NaHCO₃: stereoselective epoxidation

                  ↓

             Epoxy alcohol 7

                  ↓

KN₃, MgSO₄, H₂O: azide-mediated epoxide opening

                  ↓

               Azidodiol

                  ↓

Ms₂O, Et₃N: conversion of both hydroxyl groups into mesylates

                  ↓

          Azido dimesylate 8

                  ↓

PPh₃: azide reduction and first intramolecular cyclization

                  ↓

      Aziridinylphosphonium salt intermediate 9

                  ↓

KOtBu: promotion of the second intramolecular substitution/cyclization

                  ↓

N-Boc-1,4-diazatricyclo[4.1.0.0²,⁷]heptane 10

In this sequence, meta-chloroperoxybenzoic acid (mCPBA) is first used for stereoselective epoxidation of the alkene, establishing the relative stereochemical relationship required for the subsequent cyclization steps. Potassium azide (KN₃) then opens the epoxide, introducing an additional nitrogen source and forming an azidodiol.

Next, methanesulfonic anhydride (Ms₂O) converts the two hydroxyl groups into mesylates, thereby transforming them into suitable leaving groups for intramolecular nucleophilic substitution.

Finally, triphenylphosphine (PPh₃) promotes reduction of the azide and formation of an aziridinylphosphonium salt intermediate through the first intramolecular substitution. Potassium tert-butoxide (KOtBu) then promotes a second intramolecular cyclization, forming a new C—N bond and completing construction of the azatricyclic scaffold.

The central logic of this sequence is to establish the relative positions of the reactive sites through epoxidation and azide-mediated epoxide opening, and then convert these functional groups into a tricyclic structure containing a highly strained C—N bond through two consecutive intramolecular substitutions.

The reported results showed that several key intermediates could be prepared on an enlarged scale. The target tricyclic precursor could be purified by sublimation under reduced pressure, its structure was confirmed by single-crystal X-ray diffraction, and it displayed a certain degree of storage stability at low temperature. These findings indicate that the precursor can feasibly be prepared, isolated, stored, and used in subsequent laboratory reactions.

3 How Strain-Release Reactions Enable Bridge-Position Functionalization

3.1 Mechanism of TsCl-Mediated Strain Release

The researchers used the reaction between 1-azatricyclo[4.1.0.0²,⁷]heptane and p-toluenesulfonyl chloride (TsCl) as a model and analyzed the transformation of the highly strained C—N bond using density functional theory calculations.[1]

A simplified reaction pathway is shown below:

1-Azatricyclo[4.1.0.0²,⁷]heptane + TsCl

                    ↓

The nitrogen atom attacks the sulfur center of TsCl,

forming an N—S bond

                    ↓

            Aziridinium intermediate

                    ↓

C—N bond cleavage and C—Cl bond formation occur concertedly

                    ↓

          Chloro-substituted azabicycloalkane

For this TsCl model system, the calculations indicated that N—S bond formation is the step with the highest energy barrier. The calculated pathway then supports concerted cleavage of the highly strained C—N bond and formation of a new C—Cl bond, opening the three-membered nitrogen-containing ring and converting the tricyclic precursor into a chloro-substituted azabicyclic product.

This process indicates that TsCl first activates the nitrogen atom of the tricyclic precursor, after which strain release drives directional transformation of the C—N bond. The calculated pathway suggests that the transformation is not simply an unselective decomposition of the tricyclic structure, but is more consistent with an ordered sequence involving nitrogen activation, C—N bond cleavage, and formation of a new chemical bond.

3.2 What Types of Structures Can Be Introduced from a Common Precursor?

Using different reagents, the researchers converted the same class of azatricyclic precursors into a range of bridge-substituted azabicyclic products.

Scaffold construction and substituent selection are therefore divided into two relatively independent stages: the tricyclic precursor is prepared first, after which the bridge-position functional group is selected according to the needs of the study. For medicinal-chemistry programs requiring rapid establishment of structure–activity relationships, this common-precursor strategy may reduce the need to develop a separate complete route for every substituted analogue and thereby improve the efficiency of analogue-library construction.

4 How the Spatial Orientation of Bridge-Position Substituents Is Controlled

The identity of a functional group determines its chemical properties, whereas the direction in which it extends from the bridged scaffold determines the three-dimensional region it occupies. The study not only enables the introduction of different functional groups at the bridge carbon, but also provides access to both endo and exo configurations, allowing substituents with different spatial orientations to be compared.

4.1 What Do Endo and Exo Mean?

In the 7-substituted 6-azabicyclo[3.1.1]heptanes and 3,6-diazabicyclo[3.1.1]heptanes discussed here, the carbon atom at position 7 and the atom at position 6 constitute two separate one-atom bridges.

The orientation of a substituent at position 7 can be described as follows:

Endo configuration: the substituent points toward the concave face of the bicyclic scaffold and approaches the other one-atom bridge;

Exo configuration: the substituent points away from the concave face of the scaffold and extends toward the exterior of the bicyclic structure.

The endo and exo products have the same atomic composition and connectivity, but their substituents project into different three-dimensional regions. They may therefore establish different spatial contacts within a target-binding site.

4.2 Direct Strain Release Primarily Affords Endo Products

Stereoselective transformation of the highly strained C—N bond in the azatricyclic precursor mainly affords endo-substituted azabicyclic products. By changing the strain-release reagent or nucleophile, halogen-, carbon-, oxygen-, sulfur-, selenium-, and nitrogen-containing functional groups can be introduced at the same bridge carbon.

The general transformation can be represented as follows:

Azatricyclic precursor + functional-group source

→ C—N bond strain release → endo-7-substituted azabicycle

The endo-7-bromoazabicycle can also serve as a common intermediate for subsequent functional-group transformations:

endo-7-Bromoazabicycle

→ stereoretentive substitution → endo-7-fluoro, hydroxy, or nitro product

The paper proposes that these reactions may first involve departure of bromide assisted by participation of the neighboring nitrogen atom, followed by nucleophilic attack. The overall process results in retention of the relative configuration at the bridge carbon. Consequently, after bromine is replaced, the newly introduced functional group retains the endo orientation.

The products shown in Figure 3 of the paper are all reported with diastereomeric ratios (dr) greater than 20:1, indicating high relative stereoselectivity in the corresponding reactions. These results show that the reactions can control the endo or exo orientation of bridge-position substituents, but they do not by themselves establish a general enantioselective synthetic method.

4.3 Accessing Exo Products through Light-Driven Metal Catalysis

Direct strain release primarily generates endo-substituted products. To obtain products in which the substituent points in the opposite, exo direction, the researchers used endo-7-bromoazabicycles as intermediates and developed two classes of visible-light-driven metal-catalyzed reactions.

Under blue-light irradiation in the presence of a photocatalyst and a nickel catalyst, the nickel-catalyzed reaction replaces the bridge-position bromine atom with an aryl or heteroaryl group, forming a new C—C bond at position 7.

The copper-catalyzed reaction forms a C—N bond at the bridge position under visible-light conditions and allows the introduction of several nitrogen-containing functional groups.

Both reaction classes generate the target products with high exo selectivity. Thus, the same carbon atom at position 7 can provide an endo-substituted product through direct strain release or be converted through a bridge-position brominated intermediate into an exo-substituted product, enabling selective control over the spatial orientation of the substituent.

Accordingly, this synthetic approach can independently control three structural variables:

5 What Was Demonstrated in the Orexin-Receptor Study?

5.1 Beginning with Scaffold Replacement

Orexin receptors (OXRs) include orexin receptor type 1 (OX1R) and orexin receptor type 2 (OX2R), both of which are G protein-coupled receptors (GPCRs). The marketed drug suvorexant is a dual orexin-receptor antagonist whose structure contains a 1,4-diazepane ring that connects two principal aromatic fragments.

Using computer-aided drug design (CADD), the researchers replaced the diazepane ring with a more rigid 3,6-diazabicyclo[3.1.1]heptane, producing compound 67. This compound displayed relatively high OX2R activity in a GPCR cell-based functional assay.

Scaffold replacement alone only indicates that this bridged aza ring can reproduce part of the geometric role of the original linker ring. A more important aspect of the study was the subsequent modification of both the substituent and its orientation at the bridge carbon at position 7.

5.2 Bridge-Position Modification Demonstrates Its Value for Structure–Activity Relationship Studies

Structure–activity relationship (SAR) studies showed that substituents at position 7 had different effects on activity and receptor-subtype selectivity.

Changes to the substituent at the bridge carbon at position 7 affected both orexin-receptor activity and OX1R/OX2R subtype selectivity. In particular, 7-endo-fluoro substitution increased receptor activity, whereas 7-endo-aniline substitution altered receptor-subtype selectivity.

These results indicate that the one-carbon bridge does more than merely constrain molecular conformation; its bridge carbon can also serve as a medicinal-chemistry modification site. By using a common tricyclic precursor to vary the bridge-position substituent and its spatial orientation, preliminary structure–activity relationships can be established around the same bridged aza scaffold.

The study demonstrates the potential utility of bridge-position functionalization in lead optimization, but the results are not sufficient to conclude that a new orexin-receptor drug candidate has been obtained.

6 How Conformational Restriction Can Be Used in Bioisosteric Design

The researchers also used 6-azabicyclo[3.1.1]heptane to constrain the conformation of azetidine and designed two representative structural examples.

The first example was based on the binding conformation of the Janus kinase (JAK) inhibitor baricitinib. The researchers synthesized compound 79 containing a 6-azabicyclo[3.1.1]heptane unit, in which the 7-exo substituent maintained an axial geometric relationship similar to that of the corresponding fragment in baricitinib.

The second example was based on the investigational anticancer molecule RRx-001. Two nitro groups were introduced at position 7 of a 6-azabicyclo[3.1.1]heptane to afford bridged analogue 80, allowing the key functional groups to retain a spatial arrangement similar to that in the parent molecule.

These two examples primarily demonstrate that the target structures can be synthesized and that a specific geometry can be obtained through bridging. The paper did not report systematic comparisons of the biological activities of compounds 79 and 80 with those of the corresponding parent molecules. It therefore cannot be concluded that the bridged analogues possess higher activity or superior drug-like properties.

The effect of conformational restriction depends on whether the fixed geometry approximates the true bioactive conformation. If the constrained orientation is correct, it may reduce the conformational adjustment required when the molecule binds to its target. If the orientation is incorrect, however, the rigid structure may instead reduce binding affinity.

7 Contributions of the Study and Remaining Questions

7.1 Core Contributions

The central contribution of this study is the use of isolable and storable azatricycloalkanes as common precursors for selective transformation of highly strained C—N bonds, thereby enabling bridge-position functionalization and control over the spatial orientation of substituents in bridged aza scaffolds.

The method primarily addresses three challenges:

① It converts a one-carbon bridge position that is normally difficult to modify at a late stage into a clearly defined reaction site;

② It enables the introduction of halogen-, carbon-, oxygen-, sulfur-, selenium-, and nitrogen-containing functional groups from a common tricyclic precursor;

③ It provides access to both endo- and exo-substituted azabicyclic products through different reaction pathways.

The study therefore provides not only a method for constructing bridged aza scaffolds, but also a means of using the one-carbon bridge as an additional site for structure–activity relationship studies and lead-compound optimization.

7.2 Questions That Remain to Be Addressed

Further application of this method will require examination of the following questions:

① Can the strategy be extended to a broader range of ring sizes, nitrogen-atom positions, and more complex substitution patterns?

② In complex molecules containing multiple sensitive functional groups, will the method retain sufficient functional-group compatibility, chemoselectivity, and stereoselectivity?

③ Can a general asymmetric synthetic method be developed to provide bridge-substituted products directly in high enantiomeric purity?

④ Can the multistep synthesis of the tricyclic precursors, together with the use of azides, organolithium reagents, and related reagents, satisfy the requirements of larger-scale preparation in terms of safety, cost, and operational robustness?

⑤ Can changes in in vitro activity resulting from bridge-position modification be translated into improvements in absorption, distribution, metabolism, excretion, pharmacokinetics, and in vivo efficacy?

The answers to these questions will determine whether this approach can progress from a novel synthetic strategy into a broadly applicable method for preparing bridged aza scaffolds in drug discovery.

8 Representative Chemical Classification Tables for Azatricycloalkane Strain Release and Functionalization of Three-Dimensional Drug Scaffolds

Table 1. Reagents for Tricyclic Precursor Construction and Ring Closure

Table 2. Reagents for Strain Release and Bridge-Position Functionalization

Table 3. Components for Light-Driven Metal Catalysis and Stereodivergent Reactions

Table 4. Functional-Group Sources and Coupling Partners

Table 5. Reagents for the Preparation of Specialized Reagents and Auxiliary Reactions

Note: The products listed above are representative Aladdin products related to scientific research and formulation studies. Additional specifications, grades, and certificate of analysis information can be retrieved from the Aladdin website using the product name, CAS number, or catalog number.

References

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[4] Meanwell N A, Loiseleur O. Applications of Isosteres of Piperazine in the Design of Biologically Active Compounds: Part 2. Journal of Agricultural and Food Chemistry, 2022, 70(36): 10972–11004. doi:10.1021/acs.jafc.2c00729.

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[7] Dow N W, Cabré A, MacMillan D W C. A General N-Alkylation Platform via Copper Metallaphotoredox and Silyl Radical Activation of Alkyl Halides. Chem, 2021, 7(7): 1827–1842. doi:10.1016/j.chempr.2021.05.005.

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